Printed circuit board heater for an amplification module

ABSTRACT

An apparatus includes a substrate, a first heating element, and a second heating element. The substrate includes a first portion, a second portion, and a third portion that is between the first portion and the second portion. The first portion is characterized by a first thermal conductivity, the second portion is characterized by a second thermal conductivity, and the third portion is characterized by a third thermal conductivity. The third thermal conductivity is less than the first thermal conductivity and the second thermal conductivity. The first heating element is coupled to the first portion of the substrate, and is configured to produce a first thermal output. The second heating element is coupled to the second portion of the substrate, and configured to produce a second thermal output. The second thermal output is different from the first thermal output.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 62/326,289, entitled “Segmented Heater for a PCRModule,” filed Apr. 22, 2016, which is incorporated herein by referencein its entirety.

BACKGROUND

The embodiments described herein relate to devices and methods formolecular diagnostic testing. More particularly, the embodimentsdescribed herein relate to heaters and methods of heating a samplevolume to amplify a nucleic acid in a molecular diagnostic testingdevice.

There are over one billion infections in the U.S. each year, many ofwhich are treated incorrectly due to inaccurate or delayed diagnosticresults. Many known point of care (POC) tests have poor sensitivity(30-70%), while the more highly sensitive tests, such as those involvingthe specific detection of nucleic acids or molecular testing associatedwith a pathogenic target, are only available in laboratories. Thus,molecular diagnostics testing is often practiced in centralizedlaboratories. Known devices and methods for conducting laboratory-basedmolecular diagnostics testing, however, require trained personnel,regulated infrastructure, and expensive, high throughputinstrumentation. Known high throughput laboratory equipment generallyprocesses many (96 to 384 and more) samples at a time, therefore centrallab testing is often done in batches. Known methods for processing testsamples typically include processing all samples collected during a timeperiod (e.g., a day) in one large run, resulting in a turn-around timeof many hours to days after the sample is collected. Moreover, suchknown instrumentation and methods are designed to perform certainoperations under the guidance of a skilled technician who adds reagents,oversees processing, and moves sample from step to step. Thus, althoughknown laboratory tests and methods are very accurate, they often takeconsiderable time, and are very expensive.

There are limited testing options available for testing done at thepoint of care (“POC”), or in other locations outside of a laboratory.Known POC testing options are often single analyte tests with lowanalytical quality. These tests are used alongside clinical algorithmsto assist in diagnosis, but are frequently verified by higher quality,laboratory tests for the definitive diagnosis. Thus, in many instances,neither consumers nor physicians are enabled to achieve a rapid,accurate test result in time to “test and treat” in one visit. As aresult, doctors and patients often determine a course of treatmentbefore they know the diagnosis. This has tremendous ramifications:antibiotics are either not prescribed when needed, leading toinfections; or antibiotics are prescribed when not needed, leading tonew antibiotic-resistant strains in the community. Moreover, knownsystems and methods often result in diagnosis of severe viralinfections, such as H1N1 swine flu, too late, limiting containmentefforts. In addition, patients lose time in unnecessary, repeated doctorvisits.

Moreover, many known POC diagnostic devices employ test strips or othersimple detection mechanisms, and often do not amplify the targetorganism. Although recent advances in technology have enabled thedevelopment of “lab on a chip” devices, such devices are often notoptimized for point-of-care testing. For example, some known devices andmethods require continuous power usage to thermally cycle the sample,which can limit the ability to produce a portable or “in home” test.Other known devices include cumbersome resistance heaters and heatsinking arrangements that are not conducive to portable or “in home”tests. Specifically, such devices can have high power usage and can betoo large to be reasonably packaged for use as a POC test.

Moreover, many known “lab on a chip” devices amplify a very small volumeof sample (e.g., less than one microliter), and are therefore not suitedfor analyzing for multiple different indications (e.g., a 3-plex or4-plex test).

Reducing the package size of a diagnostic device can also negativelyimpact the accuracy to which amplification temperatures are controlled.For example, the size, shape and packaging of certain structures (e.g.,a heater, a flow member, or the like) can cause spatial variations intemperature within a reaction chamber. Moreover, tight packaging ofelectrical components (e.g., resistance heaters) and processors (e.g.,control modules) can result in an undesirable level of electromagneticfield (EMF) noise.

Thus, a need exists for improved devices and methods for moleculardiagnostic testing. In particular, a need exists for improvedamplification modules, heaters and methods for amplifying a targetnucleic acid in a molecular diagnostic testing device.

SUMMARY

Amplification modules and heaters for amplifying a nucleic acid within asample are described herein. In some embodiments, an apparatus includesa substrate, a first heating element, and a second heating element. Thesubstrate includes a first portion, a second portion, and a thirdportion that is between the first portion and the second portion. Thefirst portion is characterized by a first thermal conductivity, thesecond portion is characterized by a second thermal conductivity, andthe third portion is characterized by a third thermal conductivity. Thethird thermal conductivity is less than the first thermal conductivityand the second thermal conductivity. The first heating element iscoupled to the first portion of the substrate, and is configured toproduce a first thermal output. The second heating element is coupled tothe second portion of the substrate, and configured to produce a secondthermal output. The second thermal output is different from the firstthermal output.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view schematic illustration of a thermalreaction module, according to an embodiment.

FIG. 2 is a perspective view schematic illustration of a thermalreaction module, according to an embodiment.

FIG. 3 is a perspective view schematic illustration of a thermalreaction module, according to an embodiment.

FIG. 4 is a perspective view schematic illustration of a thermalreaction module, according to an embodiment.

FIG. 5 is an exploded perspective view of an amplification module,according to an embodiment.

FIG. 6 is an exploded view of a flow member of the amplification moduleshown in FIG. 5.

FIG. 7 is a top view of the flow member of the amplification moduleshown in FIG. 6.

FIG. 8 is a perspective view of a heater assembly of the amplificationmodule shown in FIG. 5.

FIGS. 9 and 10 are exploded perspective views of the heater assemblyshown in FIG. 8.

FIG. 11 is a top view of the heater assembly show in FIG. 8, showing afirst (or top) layer of the assembly.

FIG. 12 is a top view of the heater assembly show in FIG. 8, showing asecond layer of the assembly.

FIG. 13 is a top view of the heater assembly show in FIG. 8, showing athird layer of the assembly.

FIG. 14 is a top view of the heater assembly show in FIG. 8, showing afourth (or bottom) layer of the assembly.

FIG. 15 is a side cross-sectional view of the of the amplificationmodule shown in FIG. 5 taken along the line X-X in FIG. 5.

FIG. 16 is a bottom perspective view of the amplification module shownin FIG. 5 coupled to a detection module, according to an embodiment.

FIG. 17 is an exploded view of the amplification module and thedetection module shown in FIG. 16.

FIG. 18 is a schematic illustration of a molecular diagnostic device,according to an embodiment.

FIG. 19 is a perspective view of a molecular diagnostic device,according to an embodiment.

FIG. 20 is a perspective view of a molecular diagnostic device shown inFIG. 19 with a portion of the housing removed.

FIG. 21 is an exploded perspective view of a detection module and anamplification module of the molecular diagnostic device shown in FIGS.19 and 20.

FIG. 22 is an exploded perspective view of the amplification moduleshown in FIG. 21.

FIG. 23 is a top view of a printed circuit board of the amplificationmodule shown in FIG. 21.

FIG. 24 is an exploded perspective view of the printed circuit boardshown in FIG. 23.

FIG. 25 is a bottom view of a heater layer of the printed circuit boardshown in FIG. 23.

FIG. 26 shows a flow chart of a method of performing sampleamplification, according to an embodiment.

FIG. 27 shows a flow chart of a method of performing a thermal processon a sample, according to an embodiment.

FIG. 28 shows a flow chart of a method of performing a thermal processon a sample, according to an embodiment.

DETAILED DESCRIPTION

In some embodiments, an apparatus is configured for a disposable,portable, single-use, inexpensive, molecular diagnostic approach. Theapparatus can include one or more modules configured to perform highquality molecular diagnostic tests, including, but not limited to,sample preparation, nucleic acid amplification (e.g., via polymerasechain reaction, isothermal amplification, or the like), and detection.In some embodiments, sample preparation can be performed by isolatingthe target pathogen/entity and removing unwanted amplification (e.g.,PCR) inhibitors. The target entity can be subsequently lysed to releasetarget nucleic acid for amplification. A target nucleic acid in thetarget entity can be amplified with a polymerase undergoing temperaturecycling or via an isothermal incubation to yield a greater number ofcopies of the target nucleic acid sequence for detection.

In some embodiments, an amplification module includes a printed circuitboard upon which a series of heaters is lithographically produced. Theamplification module further includes a flow member coupled to theprinted circuit board through which a sample is conveyed to amplify atarget nucleic acid within the sample. The amplification can beperformed, for example, by cycling the sample between varioustemperature set points, by maintaining the sample at a desiredtemperature (e.g., isothermal methods), or any other suitable method. Insome embodiments, the printed circuit board also includes a processor orcontrol module.

In some embodiments, an amplification module includes a substrate, afirst heating element, and a second heating element. The substratedefines an aperture that separates the substrate into a first portionand a second portion. The first heating element is coupled to the firstportion of the substrate, and is configured to produce a first thermaloutput. The second heating element is coupled to the second portion ofthe substrate, and configured to produce a second thermal output. Thesecond thermal output is different from the first thermal output.

In some embodiments, an amplification module includes a substrate, afirst heating element, and a second heating element. The substrateincludes a first portion, a second portion, and a third portion that isbetween the first portion and the second portion. The first portion ischaracterized by a first thermal conductivity, the second portion ischaracterized by a second thermal conductivity, and the third portion ischaracterized by a third thermal conductivity. The third thermalconductivity is less than the first thermal conductivity and the secondthermal conductivity.

In some embodiments, an amplification module includes a flow member, asubstrate, a first heater assembly, and a second heater assembly. Theflow member defines a flow path through which a sample can flow from aninlet opening to an outlet opening. The first heater assembly is coupledbetween the substrate and the flow member. The first heater assembly isconfigured to maintain a first portion of the flow member at a firsttemperature. The first heater assembly includes a first set of heatingelements along a flow axis defined by the flow member. Each heatingelement from the first set of heating elements is electrically isolatedfrom the other heating elements from the first set of heating elements.The second heater assembly is coupled between the substrate and the flowmember. The second heater assembly is configured to maintain a secondportion of the flow member at a second temperature. The second heaterassembly includes a second set of heating elements along the flow axis.Each heating element from the second set of heating elements iselectrically isolated from the other heating elements from the secondset of heating elements.

In some embodiments, an amplification module can be included within adiagnostic device, which can be battery powered, allowing the diagnostictest(s) to be run without A/C power, and at any suitable location (e.g.,outside of a laboratory and/or at any suitable “point of care”). Inother embodiments, an amplification module, including any of the heaterassemblies described herein, can be included within a diagnostic devicethat is compact and consumes a limited amount of power, thus beingsuitable for use in lower current A/C circuits.

In some embodiments, an amplification module can be included within adiagnostic device that is optimized for disposable and portableoperation. For example, in some embodiments, an apparatus includes apower module operated by a small battery (e.g., a 9V battery). In suchembodiments, the device can include a controller to control the timingand/or magnitude of power draw to accommodate the capacity of thebattery.

In some embodiments, an amplification module can be included within adiagnostic device that is optimized for one-time use. In someembodiments, the diagnostic device is disposable via standard wasteprocedures after use.

In some embodiments, a method includes conveying a sample into adiagnostic device. The diagnostic device includes a flow member and aheater assembly. The flow member defines a flow path. The heaterassembly includes a substrate, a first heating element, and a secondheating element. The heater assembly coupled to the flow member suchthat the first heating element is between a first portion of thesubstrate and a first portion of the flow path, and the second heatingelement is between a second portion of the substrate and a secondportion of the flow path. A third portion of the substrate separates thefirst portion of the substrate and the second portion of the substrate.The third portion of the substrate is characterized by a thermalconductivity that is less than a thermal conductivity of the firstportion of the substrate. The device is then actuated to (1) supply afirst current to the first heating element such that the first heatingelement maintains the first portion of the flow path at a firsttemperature; (2) supply a second current to the second heating elementsuch that the second heating element maintains the second portion of theflow path at a second temperature that is different from the firsttemperature; and (3) produce a flow of the sample within the flow path.

In some embodiments, a method includes conveying a sample into adiagnostic device. The diagnostic device includes a flow member coupledto a first heater assembly and a second heater assembly. The flow memberdefines a flow path having a set of flow channels. The first heaterassembly includes a first heating element and a second heating element.The second heater assembly includes a third heating element and a fourthheating element. The device is then actuated to (1) supply, at a firsttime, current to the first heating element and the third heating elementsuch that the first heating element maintains at least a first portionof a first channel from the set of channels at a first temperature andthe third heating element maintains at least a second portion of thefirst channel from the set of channels at a second temperature; (2)produce, at a second time, a flow of the sample within the flow path;and (3) supply, at a third time, current to the second heating elementand the fourth heating element such that the second heating elementmaintains at least a first portion of a second channel from the set ofchannels at the first temperature and the fourth heating elementmaintains at least a second portion of the second channel from the setof channels at the second temperature. In some embodiments, the secondtime occurs after the first time, and the third time is different fromthe first time.

In some embodiments, a method includes conveying a sample into adiagnostic device. The diagnostic device includes a flow member coupledto a first heater assembly and a second heater assembly. The flow memberdefines a flow path having a plurality of flow channels. The firstheater assembly includes a first heating element, and the second heaterassembly includes a second heating element and a third heating element.The first heater assembly is coupled to the flow member such that thefirst heating element is aligned with a first portion of the flow path.The second heater assembly is coupled to the flow member such that andthe second heating element and the third heating element are eachaligned with a second portion of the flow path. The device is thenactuated to (1) supply a first current to the first heating element suchthat the first heating element maintains the first portion of the flowpath at a first temperature; (2) produce a flow of the sample within theflow path; (3) supply a second current to the second heating element;and (4) supply a third current to the third heating element. The thirdcurrent is supplied independently from the second current, and thesecond current and the third current supplied such that the secondheating element and the third heating element collectively maintain thesecond portion of the flow path at a second temperature.

As used herein, the term “about” when used in connection with areferenced numeric indication means the referenced numeric indicationplus or minus up to 10% of that referenced numeric indication. Forexample, the language “about 50” covers the range of 45 to 55.

As used in this specification and the appended claims, the words“proximal” and “distal” refer to direction closer to and away from,respectively, an operator of the diagnostic device. Thus, for example,the end of an actuator depressed by a user that is furthest away fromthe user would be the distal end of the actuator, while the end oppositethe distal end (i.e., the end manipulated by the user) would be theproximal end of the actuator.

The term “fluid-tight” is understood to encompass hermetic sealing(i.e., a seal that is gas-impervious) as well as a seal that is onlyliquid-impervious. The term “substantially” when used in connection with“fluid-tight,” “gas-impervious,” and/or “liquid-impervious” is intendedto convey that, while total fluid imperviousness is desirable, someminimal leakage due to manufacturing tolerances, or other practicalconsiderations (such as, for example, the pressure applied to the sealand/or within the fluid), can occur even in a “substantiallyfluid-tight” seal. Thus, a “substantially fluid-tight” seal includes aseal that prevents the passage of a fluid (including gases, liquidsand/or slurries) therethrough when the seal is maintained at pressuresof less than about 5 psig, less than about 10 psig, less than about 20psig, less than about 30 psig, less than about 50 psig, less than about75 psig, less than about 100 psig, and all values in between. Anyresidual fluid layer that may be present on a portion of a wall of acontainer after component defining a “substantially-fluid tight” sealare moved past the portion of the wall are not considered as leakage.

The term “parallel” is used herein to describe a relationship betweentwo geometric constructions (e.g., two lines, two planes, a line and aplane, or the like) in which the two geometric constructions arenon-intersecting as they extend substantially to infinity. For example,as used herein, a planar surface (i.e., a two-dimensional surface) issaid to be parallel to a line when every point along the line is spacedapart from the nearest portion of the surface by a substantially equaldistance. Similarly, a first line (or axis) is said to be parallel to asecond line (or axis) when the first line and the second line do notintersect as they extend to infinity. Two geometric constructions aredescribed herein as being “parallel” or “substantially parallel” to eachother when they are nominally parallel to each other, such as forexample, when they are parallel to each other within a tolerance. Suchtolerances can include, for example, manufacturing tolerances,measurement tolerances or the like.

The terms “perpendicular,” “orthogonal,” and “normal” are used herein todescribe a relationship between two geometric constructions (e.g., twolines, two planes, a line and a plane, or the like) in which the twogeometric constructions intersect at an angle of approximately 90degrees within at least one plane. For example, as used herein, a line(or axis) is said to be normal to a planar surface when the line and aportion of the planar surface intersect at an angle of approximately 90degrees within the planar surface. Two geometric constructions aredescribed herein as being, for example, “perpendicular” or“substantially perpendicular” to each other when they are nominallyperpendicular to each other, such as for example, when they areperpendicular to each other within a tolerance. Such tolerances caninclude, for example, manufacturing tolerances, measurement tolerancesor the like.

Similarly, geometric terms, such as “parallel,” “perpendicular,”“cylindrical,” “square,” “conical,” or “frusto-conical” are not intendedto require absolute mathematical precision, unless the context indicatesotherwise. Instead, such geometric terms allow for variations due tomanufacturing or equivalent functions. For example, if an element isdescribed as “conical” or “generally conical,” a component that is notprecisely conical (e.g., one that is slightly oblong) is stillencompassed by this description.

As used in this specification and the appended claims, the term“reagent” includes any substance that is used in connection with any ofthe reactions described herein. For example, a reagent can include anelution buffer, a PCR reagent, an enzyme, a substrate, a wash solution,or the like. A reagent can include a mixture of one or moreconstituents. A reagent can include such constituents regardless oftheir state of matter (e.g., solid, liquid or gas). Moreover, a reagentcan include the multiple constituents that can be included in asubstance in a mixed state, in an unmixed state and/or in a partiallymixed state. A reagent can include both active constituents and inertconstituents. Accordingly, as used herein, a reagent can includenon-active and/or inert constituents such as, water, colorant or thelike.

The term “nucleic acid molecule,” “nucleic acid,” or “polynucleotide”may be used interchangeably herein, and may refer to deoxyribonucleicacid (DNA) or ribonucleic acid (RNA), including known analogs or acombination thereof unless otherwise indicated. Nucleic acid moleculesto be profiled herein can be obtained from any source of nucleic acid.The nucleic acid molecule can be single-stranded or double-stranded. Insome cases, the nucleic acid molecules are DNA The DNA can bemitochondrial DNA, complementary DNA (cDNA), or genomic DNA. In somecases, the nucleic acid molecules are genomic DNA (gDNA). The DNA can beplasmid DNA, cosmid DNA, bacterial artificial chromosome (BAC), or yeastartificial chromosome (YAC). The DNA can be derived from one or morechromosomes. For example, if the DNA is from a human, the DNA can bederived from one or more of chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, or Y. In some cases,the nucleic acid molecules are RNA can include, but is not limited to,mRNAs, tRNAs, snRNAs, rRNAs, retroviruses, small non-coding RNAs,microRNAs, polysomal RNAs, pre-mRNAs, intronic RNA, viral RNA, cell freeRNA and fragments thereof. The non-coding RNA, or ncRNA can includesnoRNAs, microRNAs, siRNAs, piRNAs and long nc RNAs. The source ofnucleic acid for use in the devices, methods, and compositions describedherein can be a sample comprising the nucleic acid.

Unless indicated otherwise, the terms apparatus, diagnostic apparatus,diagnostic system, diagnostic test, diagnostic test system, test unit,and variants thereof, can be interchangeably used.

FIG. 1 is a schematic illustration of an amplification (or thermalreaction) module 1600, according to an embodiment. The amplificationmodule 1600 is configured to perform a thermal reaction (e.g., anamplification reaction) on an input of target DNA mixed with requiredreagents, and can be included in any suitable diagnostic device. Forexample, the amplification module can be included in any of thediagnostic devices shown and described herein (including the device 6000and the device 7000) or in International Patent Publication No.WO2016/109691, entitled “Devices and Methods for Molecular DiagnosticTesting,” which is incorporated herein by reference in its entirety. Insome embodiments, the amplification module 1600 is configured to conductrapid PCR amplification of an input target. In some embodiments, theamplification module 1600 is configured to generate an output copynumber that reaches or exceeds the threshold of the sensitivity of anassociated detection module.

The amplification module 1600 includes a flow member 1610 and a heaterassembly 1630. The flow member 1610 defines a flow path 1620 throughwhich a sample can flow from an inlet port 1621 to an outlet port 1622.The flow member 1610 defines a flow axis A_(F) that indicates theoverall direction of the flow through the flow member 1610. As describedin more detail below, the flow path 1620 is shaped and/or has a geometrysuch that various portions of the flow path 1620 (e.g., a first portion1624 and a second portion 1625) can be maintained at differenttemperatures by the heater assembly 1630. In this manner, theamplification module 1600 can perform a “flow through” polymerase chainreaction (PCR) on the sample to amplify the target organism and/orportions of a nucleic acid with within the sample. Although the flowpath 1620 is shown as being a serpentine path (or a path that includesmultiple switchbacks to reverse the flow of the sample), in otherembodiments, the flow path can have any suitable shape and/or geometry.

The flow member 1610 (and any of the flow members described herein) canbe constructed from any suitable material and can have any suitabledimensions to facilitate the desired amplification performance for thedesired volume of sample. For example, in some embodiments, theamplification module 1600 (and any of the amplification modulesdescribed herein) can perform 1000× or greater amplification in a timeof less than 15 minutes. For example, in some embodiments, the flowmember 1610 (and any of the flow members described herein) isconstructed from at least one of a cyclic olefin copolymer or agraphite-based material. Such materials facilitate the desired heattransfer properties into the flow path 1620. Moreover, in someembodiments, the flow member 1610 (and any of the flow members describedherein) can have a thickness of less than about 0.5 mm. In someembodiments, the flow member 1610 (and any of the flow members describedherein) can have a volume about 150 microliters or greater, and the flowcan be such that at least 10 microliters of sample is amplified. Inother embodiments, at least 20 microliters of sample are amplified bythe methods and devices described herein. In other embodiments, at least30 microliters of sample are amplified by the methods and devicesdescribed herein. In yet other embodiments, at least 50 microliters ofsample are amplified by the methods and devices described herein.

The heater assembly 1630 includes a substrate 1640, a first heatingelement 1650, and a second heating element 1660, each coupled to thesubstrate 1640. As described herein, the heater assembly 1630 is coupledto the flow member 1610, and is configured to maintain various portionsof the flow path 1620 at different temperature set points to facilitatethe desired reaction. Thus, the substrate 1640 can be any suitablesubstrate, such as for example, an electrically isolative substrate towhich the first heating element 1650 and second heating element 1660 aremounted. Moreover, the substrate 1640 (and any of the substratesdescribed herein) can be constructed from any suitable material, suchas, for example, a composite material including woven glass and epoxy.In some embodiments, the substrate 1640 (and any of the substratesdescribed herein) can be constructed from a material having a glasstransition temperature (Tg) of greater than about 170 C. In otherembodiments, the substrate 1640 (and any of the substrates describedherein) can be constructed from a material having a glass transitiontemperature (Tg) of greater than about 180 C (e.g., material 370HRproduced by the Isola Group). In this manner, the substrate 1640 canmaintain the desired rigidity and dimensional integrity to providerepeatable thermal performance for each channel of the flow path 1620.

As shown, the substrate 1640 defines an aperture 1641 (also referred toas an opening, cut-out, or via) that separates the substrate 1640 into afirst portion 1631 and a second portion 1632. The first heating element1650 is coupled to the first portion 1631, and the second heatingelement 1660 is coupled to the second portion 1632. In this manner, theaperture 1641 thermally isolates the first portion 1631 from the secondportion 1632. Thus, by minimizing the heat transfer between the firstportion 1631 and the second portion 1632, accuracy of the heat flow fromthe first heating element 1650 and the second heating element 1660 tothe flow member 1610 can be improved. More particularly, the substrate1640 is coupled to the flow member 1610 such that the first portion 1631of the substrate 1640 is aligned with the first portion 1624 of the flowpath 1620 and such that the second portion 1632 of the substrate 1640 isaligned with the second portion 1625 of the flow path 1620. Thisarrangement allows the first heating element 1650 to heat the firstportion 1624 of the flow path 1620, and the second heating element 1660to heat the second portion 1625 of the flow path 1620.

In use, the first heating element 1650 produces a first thermal outputQ₁ to maintain the first portion 1624 of the flow path 1620 at a firsttemperature. The first temperature can be, for example, between about100 C and about 115 C (to heat the sample to about 90 C; e.g., the “hot”temperature for a PCR thermal cycle). The second heating element 1660produces a second thermal output Q₂ that is different from the firstthermal output Q₁, and that can maintain the second portion 1625 of theflow path 1620 at a second temperature. The second temperature can be,for example, between about 60 C and about 75 C (to heat the sample toabout 60 C; e.g., the “cold” temperature for a PCR thermal cycle). Inthis manner, the heater assembly 1630 and the flow member 1610 canestablish multiple temperature zones through which a sample can flow,and can define a desired number of amplification cycles to ensure thedesired test sensitivity (e.g., at least 30 cycles, at least 34 cycles,at least 36 cycles, at least 38 cycles, or at least 40 cycles).

In some embodiments, the sample flowing within the flow path 1620 israpidly heated to about 90 C. To promote a rapid cooling down to about60 C, in some embodiments, heat must flow out of the sample (and thusthe flow member 1610). Thus, although the second thermal output Q₂ isshown as flowing into the flow path 1620 and/or the flow member 1610, inother embodiments, the second thermal output produced by the secondheating element (or any of the heating elements described herein) can besuch that the second thermal output Q₂ flows out of the flow path 1620and/or the flow member 1610 towards the second heating element 1660. Insuch embodiments, a current can still be supplied to the second heatingelement 1660 to control the magnitude of the heat flow. In someembodiments, the second temperature can be, for example, between about40 C and about 45 C (to allow heat transfer away from the sample at acontrolled rate to facilitate maintaining the sample at about 60 C;e.g., the “cold” temperature for a PCR thermal cycle).

The aperture 1641 defined by the substrate 1640 can be of any suitablesize and/or shape to facilitate thermal isolation of the first portion1631 of the substrate 1640 and the second portion 1632 of the substrate1640. For example, as shown in FIG. 1, the aperture 1641 can be anelongated opening that extends (or is elongated) along the overalldirection of flow, as indicated by the flow axis A_(F). In otherembodiments, however, the aperture 1641 or portions thereof can bealigned with the channels of the flow path 1620. Similarly stated, insome embodiments, the aperture 1641 need not be a linear aperture, butcan instead have multiple sections that are lateral to the flow axisA_(F). In yet other embodiments, the aperture 1641 can be one of aseries of openings, cut-outs, or slots that collectively thermallyisolate the first portion 1631 of the substrate 1640 from the secondportion 1632 of the substrate 1640. In still other embodiments, theaperture 1641 need not be a through-opening, but rather can be a blindopening that does not extend entirely through the substrate 1640.

The first heating element 1650 and the second heating element 1660 canbe any suitable heating element or collection of heaters that canperform the functions described herein. For example, in someembodiments, the first heating element 1650 and the second heatingelement 1660 can each be single heating element that is thermallycoupled to the flow member 1610, and that can cycle through multipletemperatures set points (e.g., between about 60 C and about 90 C).Moreover, the first heating element 1650 and the second heating element1660 can be of any suitable design. For example, in some embodiments,the first heating element 1650 and the second heating element 1660 canbe a resistance heater, a thermoelectric device (e.g. a Peltier device),or the like. In some embodiments, the first heating element 1650 and thesecond heating element 1660 can be resistance heaters that arelithographically produced on or within the substrate 1640.

The flow member 1610 can be coupled to the heater assembly 1630 in anysuitable manner. For example, in some embodiments, the flow member 1610can be coupled to the heater assembly 1630 by a series of mechanicalfasteners, such as clamps, screws, or the like. In some suchembodiments, the fasteners can also function as heat sinks to allowaccurate control of the temperatures of the flow member 1610 and toavoid overheating. In other embodiments, the flow member 1610 can becoupled to the heater assembly 1630 by an adhesive (e.g., apressure-sensitive adhesive). Similarly stated, in some embodiments, theflow member 1610 can be chemically bonded to the heater assembly 1630.In yet other embodiments, the flow member 1610 can be coupled to theheater assembly 1630 by an adhesive (e.g., a pressure-sensitiveadhesive) and mechanical fasteners can be used to couple other structureand function as a heat sink. In this manner, the flow member 1610 isfixedly coupled to the heater assembly 1630. Said another way, in someembodiments, the flow member 1610 is not designed to be removed and/ordecoupled from the heater assembly 1630 during normal use (i.e., theflow member 1610 is irreversibly coupled to the heater assembly 1630and/or the substrate 1640). This arrangement facilitates a single-use,disposable device that includes the amplification module 1600.

Although the substrate 1640 is shown as defining an aperture 1641, insome embodiments, an amplification module can include a substrate thatdoes not define an aperture that separates heaters mounted thereto.Moreover, although the flow member 1610 is shown as defining aserpentine flow path 1620, in other embodiments, an amplification modulecan include any suitably-shaped flow path. For example, FIG. 2 is aschematic illustration of an amplification (or thermal reaction) module2600, according to an embodiment. The amplification module 2600 isconfigured to perform a thermal reaction (e.g., an amplificationreaction) on an input of target nucleic acid mixed with requiredreagents, and can be included in any suitable diagnostic device. Forexample, the amplification module can be included in any of thediagnostic devices shown and described herein (including the device 6000and the device 7000) or in International Patent Publication No.WO2016/109691, entitled “Devices and Methods for Molecular DiagnosticTesting,” which is incorporated herein by reference in its entirety. Insome embodiments, the amplification module 2600 is configured to conductrapid amplification of an input target. In some embodiments, theamplification module 2600 is configured to generate an output copynumber that reaches or exceeds the threshold of the sensitivity of anassociated detection module.

The amplification module 2600 includes a flow member 2610 and a heaterassembly 2630. The flow member 2610 defines a flow path 2620 throughwhich a sample can flow from an inlet port to an outlet port. The flowmember 2610 defines a flow axis A_(F) that indicates the overalldirection of the flow through the flow member 2610. The flow path 2620includes a first portion 2624, a second portion 2625, and a thirdportion 2626. Similarly stated, the walls of the flow member define thefirst portion 2624, the second portion 2625, and the third portion 2626,which collectively form the flow path 2620. As described herein, thevarious portions of the flow path 2620 can be maintained at differenttemperatures by the heater assembly 2630. In this manner, theamplification module 2600 can perform a variety of thermally-basedoperations on a sample within the flow member 2610. For example, in someembodiments, a sample within the flow member 2610 can be repeatedlymoved between the first portion 2624 and the second portion 2625 tothermally cycle the sample. In this manner, the amplification module2600 can be used to perform a polymerase chain reaction (PCR) on thesample to amplify a target organism and/or portions of a nucleic acidwith within the sample. In other embodiments, a sample within the flowmember 2610 can be maintained at a substantially constant temperaturewithin the first portion 2624 and/or the second portion 2625 to performan isothermal amplification process to amplify a target organism and/orportions of a nucleic acid with within the sample. In yet otherembodiments, a sample within the flow member 2610 can be maintained at afirst temperature within the first portion 2624 and a second temperaturewith the second portion 2625. In this manner, the amplification module2600 can be used to perform multiple operations on the sample (e.g., alysing and/or inactivation operation within the first portion 2624followed by an isothermal amplification operation within the secondportion 2625). Although the flow path 2620 is shown as beingsubstantially linear, in other embodiments, the flow path can have anysuitable shape and/or geometry.

The flow member 2610 (and any of the flow members described herein) canbe constructed from any suitable material and can have any suitabledimensions to facilitate the desired amplification performance for thedesired volume of sample. For example, in some embodiments, theamplification module 2600 (and any of the amplification modulesdescribed herein) can perform 1000× or greater amplification in a timeof less than 15 minutes. For example, in some embodiments, the flowmember 2610 (and any of the flow members described herein) isconstructed from at least one of a cyclic olefin copolymer or agraphite-based material.

Such materials facilitate the desired heat transfer properties into theflow path 2620. Moreover, in some embodiments, the flow member 2610 (andany of the flow members described herein) can have a thickness of lessthan about 0.5 mm. In some embodiments, the flow member 2610 (and any ofthe flow members described herein) can have a volume about 150microliters or greater, and the flow can be such that at least 10microliters of sample is amplified. In other embodiments, at least 20microliters of sample are amplified by the methods and devices describedherein. In other embodiments, at least 30 microliters of sample areamplified by the methods and devices described herein. In yet otherembodiments, at least 50 microliters of sample are amplified by themethods and devices described herein.

The heater assembly 2630 includes a substrate 2640, a first heatingelement 2650, and a second heating element 2660 each coupled to thesubstrate 2640. As described herein, the heater assembly 2630 can becoupled to the flow member 2610, and is configured to maintain variousportions of the flow path 2620 at different temperature set points tofacilitate the desired reaction (e.g., a thermal cycling amplification,an isotherm amplification, a lysis reaction, or the like). Thus, thesubstrate 2640 can be any suitable substrate, such as for example, anelectrically isolative substrate to which the first heating element 2650and second heating element 2660 are mounted. Moreover, the substrate2640 (and any of the substrates described herein) can be constructedfrom any suitable material, such as, for example, a composite materialincluding woven glass and epoxy. In some embodiments, the substrate 2640(and any of the substrates described herein) can be constructed from amaterial having a glass transition temperature (Tg) of greater thanabout 170 C. In other embodiments, the substrate 2640 (and any of thesubstrates described herein) can be constructed from a material having aglass transition temperature (Tg) of greater than about 180 C (e.g.,material 370HR produced by the Isola Group). In this manner, thesubstrate 2640 can maintain the desired rigidity and dimensionalintegrity to provide repeatable thermal performance for each portion ofthe flow path 2620.

In some embodiments, the heater assembly 2630 and the substrate 2640(and any of the heater assemblies and substrates described herein) canbe a portion of a printed circuit board of a molecular diagnosticsdevice (e.g., any of the devices described herein, including the device6000 and the device 7000). Thus, the substrate 2640 can also supportand/or be coupled to electronic components that form the circuitry tocontrol the heater assembly 2630 as well as the overall diagnosticdevice (e.g., flow pumps, introduction of reagents, sample preparationoperations, or the like). For example, in some embodiments, thesubstrate 2640 (and any of the substrates or printed circuit boardlayers described herein) can be coupled to and/or support a processor, acontroller, or the like. In this manner, the heater assembly 2630 andthe substrate 2640 (and any of the heater assemblies and substratesdescribed herein) can be a portion of a printed circuit board thatperforms many different electronic functions, including controlling theamplification of the sample, controlling sample movement, and otherthermally-based functions described herein.

As shown, the substrate 2640 includes a first portion 2631, a secondportion 2632, and a third portion 2633. The third portion 2633 isbetween the first portion 2631 and the second portion 2632. Similarlystated, the third portion 2633 separates the first portion 2631 and thesecond portion 2632. The first heating element 2650 is coupled to thefirst portion 2631 of the substrate 2640 and can produce a first thermaloutput Q₁. The second heating element 2660 is coupled to the secondportion 2632 of the substrate 2640 and can produce a second thermaloutput Q₂. In some embodiments, the substrate 2640 can be coupled to theflow member 2610 such that the first portion 2631 of the substrate 2640(and the first heating element 2650) is aligned with the first portion2624 of the flow path 2620 and the second portion 2632 of the substrate2640 (and the second heating element 2660) is aligned with the secondportion 2625 of the flow path 2620. This arrangement allows the firstheating element 2650 to heat the first portion 2624 of the flow path2620, and the second heating element 2660 to heat the second portion2625 of the flow path 2620, as described herein.

The first portion 2631 of the substrate 2640 is characterized by a firstthermal conductivity, the second portion 2632 of the substrate 2640 ischaracterized by a second thermal conductivity, and the third portion2633 of the substrate 2640 is characterized by a third thermalconductivity. The third thermal conductivity is less than the firstthermal conductivity and the second thermal conductivity. In thismanner, heat transfer within the substrate 2640 between the firstportion 2631 and the second portion 2632 is limited. Similarly stated,the difference in the thermal conductivity between the third portion2633 and the other two portions (i.e., the first portion 2631 and thesecond portion 2632) is such that the third portion 2633 thermallyisolates the first portion 2631 from the second portion 2632. Byminimizing the heat transfer between the first portion 2631 and thesecond portion 2632, accuracy of the heat flow from the first heatingelement 2650 and the second heating element 2660 to the flow member 2610can be improved.

In some embodiments, the third portion 2633 of the substrate 2640 can beconstructed from a different material from which either of the firstportion 2631 of the substrate 2640 or the second portion 2632 of thesubstrate 2640 are constructed. For example, in some embodiments, thefirst portion 2631 of the substrate 2640 and the second portion 2632 ofthe substrate 2640 are constructed from a composite material includingwoven glass and epoxy, including, for example, an FR-4 grade material.Such materials can have a thermal conductivity of between about 0.5W/m-K and about 1.0 W/m-K. In contrast the third portion 2633 of thesubstrate 2640 can be constructed from or include a material having alower thermal conductivity. For example, in some embodiments, the thirdportion 2633 of the substrate 2640 can be constructed from or include amaterial having a thermal conductivity of about 0.1 W/m-K or less. Inother embodiments, the third portion 2633 of the substrate 2640 can beconstructed from or include a material having a thermal conductivity ofabout 0.05 W/m-K or less. For example, in some embodiments, the thirdportion 2633 of the substrate 2640 (or any other insulative substrateportions described herein) can be constructed from or include a rigidfoam (e.g., polyurethane foam, a silicon foam, a neoprene foam, a vinylfoam, or the like). In other embodiments, the third portion 2633 of thesubstrate 2640 (or any other insulative substrate portions describedherein) can be constructed from or include a low thermal conductivitypolymer or composite material.

The third portion 2633 of the substrate 2640 can provide a lower thermalconductivity (or higher thermal resistance) than that of either of thefirst portion 2631 of the substrate 2640 or the second portion 2632 ofthe substrate 2640 along any axis. For example, in some embodiment, thethird portion 2633 of the substrate 2640 can have a lower “in plane”thermal conductivity (or higher “in plane” thermal resistance) than thatof either of the first portion 2631 of the substrate 2640 or the secondportion 2632 of the substrate 2640. In this manner, the heat flowbetween the first portion 2631 of the substrate 2640 and the secondportion 2632 of the substrate 2640 within the planar surface to whichthe heating elements are coupled is limited.

Moreover, although the first portion 2631 of the substrate 2640, thesecond portion 2632 of the substrate 2640, and the third portion 2633 ofthe substrate 2640 are shown as defining a planar surface to which theflow member 2610 is coupled, in other embodiments, the top surface ofthe substrate 2640 and/or heater assembly 2630 need not be planar. Inother embodiments, for example, the top surface of the substrate 2640can include multiple different levels and/or discontinuous portions.

In some embodiments, the third portion 2633 of the substrate 2640 candefine one or more apertures or openings (not shown in FIG. 2) todecrease the thermal conductivity (or increase the thermal resistance)of the third portion 2633. For example, in some embodiments, the thirdportion 2633 of the substrate 2640 (or any other substrate portionsdescribed herein) can include a series of perforations. Suchperforations can either extend through the substrate (i.e., “throughholes”) or can extend only partially through the substrate. Moreover, inthose embodiments in which the third portion 2633 of the substrate 2640defines one or more apertures, the substrate 2640 (or any of thesubstrates described herein) can include one or more connection portionsto maintain the desired structural rigidity of the third portion 2633.

The first heating element 2650 and the second heating element 2660 canbe any suitable heating element or collection of heaters that canperform the functions described herein. For example, in someembodiments, the first heating element 2650 and the second heatingelement 2660 can each be single heating element that is thermallycoupled to the flow member 2610, and that can cycle through multipletemperatures set points (e.g., between about 60 C and about 90 C).Moreover, the first heating element 2650 and the second heating element2660 can be of any suitable design. For example, in some embodiments,the first heating element 2650 and the second heating element 2660 canbe a resistance heater, a thermoelectric device (e.g. a Peltier device),or the like. In some embodiments, the first heating element 2650 and thesecond heating element 2660 can be resistance heaters that arelithographically produced on or within the substrate 2640.

The flow member 2610 can be coupled to the heater assembly 2630 in anysuitable manner. For example, in some embodiments, the flow member 2610can be coupled to the heater assembly 2630 by a series of mechanicalfasteners, such as clamps, screws, or the like. In some suchembodiments, the fasteners can also function as heat sinks to allowaccurate control of the temperatures of the flow member 2610 and toavoid overheating. In other embodiments, the flow member 2610 can becoupled to the heater assembly 2630 by an adhesive (e.g., apressure-sensitive adhesive). Similarly stated, in some embodiments, theflow member 2610 can be chemically bonded to the heater assembly 2630.In yet other embodiments, the flow member 2610 can be coupled to theheater assembly 2630 by an adhesive (e.g., a pressure-sensitiveadhesive) and mechanical fasteners can be used to couple other structureand function as a heat sink. In this manner, the flow member 2610 isfixedly coupled to the heater assembly 2630. Said another way, in someembodiments, the flow member 2610 is not designed to be removed and/ordecoupled from the heater assembly 2630 during normal use (i.e., theflow member 2610 is irreversibly coupled to the heater assembly 2630and/or the substrate 2640). This arrangement facilitates a single-use,disposable device that includes the amplification module 2600.

In use, the first heating element 2650 produces a first thermal outputQ₁ to maintain the first portion 2624 of the flow path 2620 (or thesample therein) at a first temperature. The first temperature can be,for example, between about 100 C and about 115 C (to heat the sample toabout 90 C; e.g., the “hot” temperature for a PCR thermal cycle). Thesecond heating element 2660 produces a second thermal output Q₂ that isdifferent from the first thermal output Q₁, and that can maintain thesecond portion 2625 of the flow path 2620 at a second temperature. Thesecond temperature can be, for example, between about 60 C and about 75C (to heat the sample to about 60 C; e.g., the “cold” temperature for aPCR thermal cycle). In this manner, the heater assembly 2630 and theflow member 2610 can establish multiple temperature zones within which asample can flow or be maintained.

In some embodiments, the sample flowing within the first portion 2624flow path 2620 is rapidly heated to about 90 C. To promote a rapidcooling down to about 60 C, in some embodiments, heat must flow out ofthe sample (and thus the flow member 2610). Thus, although the secondthermal output Q₂ is shown as flowing into the flow path 2620 and/or theflow member 2610, in other embodiments, the second thermal outputproduced by the second heating element (or any of the heating elementsdescribed herein) can be such that the second thermal output Q₂ flowsout of the flow path 2620 and/or the flow member 2610 towards the secondheating element 2660. In such embodiments, a current can still besupplied to the second heating element 2660 to control the magnitude ofthe heat flow. In some embodiments, the second temperature can be, forexample, between about 40 C and about 45 C (to allow heat transfer awayfrom the sample at a controlled rate to facilitate maintaining thesample at about 60 C; e.g., the “cold” temperature for a PCR thermalcycle).

In other embodiments, the first temperature can be maintained at atemperature suitable for cell lysis, and the second temperature can bemaintained at a temperature suitable for an isothermal amplificationoperation. In this manner, the sample (and the nucleic acid(s) therein)can be prepared for amplification in the first portion 2624 of the flowpath 2620, and then amplified in the second portion 2625 of the flowpath 2620. In some embodiments, the amplification module 2600 (and anyof the amplification modules described herein) can perform any suitabletype of isothermal amplification process, including, for example, LoopMediated Isothermal Amplification (LAMP), Nucleic Acid Sequence BasedAmplification (NASBA), which can be useful to detect target RNAmolecules, Strand Displacement Amplification (SDA), MultipleDisplacement Amplification (MDA), Ramification Amplification Method(RAM), or any other type of isothermal process.

In other embodiments, any suitable thermal reaction can be conductedeither of the first portion 2624 of the flow path 2620 or the secondportion 2625 of the flow path 2620. For example, in some embodiments,the temperature of the first portion 2624 (or any of the flow pathportions described herein) can be maintained to perform a hot-start onthe sample therein, while the temperature of the second portion 2625 canbe maintained or cycled to amplify the organisms therein. In otherembodiments, either of the first portion 2624 of the flow path 2620 orthe second portion 2625 of the flow path 2620 can be used to conduct alysing reaction, an inactivation reaction, and/or a detection reaction.

Although the substrate 2640 is shown as including one heating elementcoupled to (or within) the first portion 2631 of the substrate 2640 anda second heating element coupled to (or within) the second portion 2632of the substrate 2640, in other embodiments, any number of heatingelements can be coupled to any portion of a substrate. Moreover, in someembodiments, the heating elements can be electrically isolated from eachother to allow for independent control of each heating element. Forexample, FIG. 3 is a schematic illustration of an amplification (orthermal reaction) module 3600, according to an embodiment. Theamplification module 3600 is configured to perform a thermal reaction(e.g., an amplification reaction) on an input of target nucleic acidmixed with required reagents, and can be included in any suitablediagnostic device. For example, the amplification module can be includedin any of the diagnostic devices shown and described herein (includingthe device 6000 and the device 7000) or in International PatentPublication No. WO2016/109691, entitled “Devices and Methods forMolecular Diagnostic Testing,” which is incorporated herein by referencein its entirety. In some embodiments, the amplification module 3600 isconfigured to conduct rapid amplification of an input target. In someembodiments, the amplification module 3600 is configured to generate anoutput copy number that reaches or exceeds the threshold of thesensitivity of an associated detection module.

The amplification module 3600 includes a flow member 3610 and a heaterassembly 3630. The flow member 3610 defines a flow path 3620 throughwhich a sample can flow from an inlet port to an outlet port. The flowmember 3610 defines a flow axis A_(F) that indicates the overalldirection of the flow through the flow member 3610. The flow path 3620includes a first portion 3624, a second portion 3625, and a thirdportion 3626. Similarly stated, the walls of the flow member define thefirst portion 3624, the second portion 3625, and the third portion 3626,which collectively form the flow path 3620. As described herein, thevarious portions of the flow path 3620 can be maintained at differenttemperatures by the heater assembly 3630. In this manner, theamplification module 3600 can perform a variety of thermally-basedoperations on a sample within the flow member 3610. For example, in someembodiments, a sample the flow member 3610 can be maintained at asubstantially constant temperature within the first portion 3624 and/orthe second portion 3625 to perform an isothermal amplification processto amplify a target organism and/or portions of a nucleic acid withwithin the sample. In other embodiments, a sample within the flow member3610 can be maintained at a first temperature within the first portion3624 and a second temperature with the second portion 3625. In thismanner, the amplification module 3600 can be used to perform multipleoperations on the sample (e.g., a hot-start, a lysing and/or aninactivation operation within the first portion 3624 followed by anisothermal amplification operation within the second portion 3625).Although the first portion 3624 of the flow path 3620 is shown as beingsubstantially linear and the second portion 3625 of the flow path 3620is shown as including a series of switchbacks, in other embodiments, theflow path can have any suitable shape and/or geometry.

The flow member 3610 (and any of the flow members described herein) canbe constructed from any suitable material and can have any suitabledimensions to facilitate the desired amplification performance for thedesired volume of sample. For example, in some embodiments, theamplification module 3600 (and any of the amplification modulesdescribed herein) can perform 1000× or greater amplification in a timeof less than 15 minutes. For example, in some embodiments, the flowmember 3610 (and any of the flow members described herein) isconstructed from at least one of a cyclic olefin copolymer or agraphite-based material. Such materials facilitate the desired heattransfer properties into the flow path 3620. Moreover, in someembodiments, the flow member 3610 (and any of the flow members describedherein) can have a thickness of less than about 0.5 mm. In someembodiments, the flow member 3610 (and any of the flow members describedherein) can have a volume about 150 microliters or greater, and the flowcan be such that at least 10 microliters of sample is amplified. Inother embodiments, at least 20 microliters of sample are amplified bythe methods and devices described herein. In other embodiments, at least30 microliters of sample are amplified by the methods and devicesdescribed herein. In yet other embodiments, at least 50 microliters ofsample are amplified by the methods and devices described herein.

The heater assembly 3630 includes a substrate 3640, a first heatingelement assembly 3650, and a second heating element assembly 3660 eachcoupled to (or within) the substrate 3640. As described herein, theheater assembly 3630 can be coupled to the flow member 3610, and isconfigured to maintain various portions of the flow path 3620 atdifferent temperature set points to facilitate the desired reaction(e.g., a thermal cycling amplification, an isotherm amplification, alysis reaction, or the like). Thus, the substrate 3640 can be anysuitable substrate, such as for example, an electrically isolativesubstrate to which the first heating element 3650 and second heatingelement 3660 are mounted. Moreover, the substrate 3640 (and any of thesubstrates described herein) can be constructed from any suitablematerial, such as, for example, a composite material including wovenglass and epoxy. In some embodiments, the substrate 3640 (and any of thesubstrates described herein) can be constructed from a material having aglass transition temperature (Tg) of greater than about 170 C. In otherembodiments, the substrate 3640 (and any of the substrates describedherein) can be constructed from a material having a glass transitiontemperature (Tg) of greater than about 180 C (e.g., material 370HRproduced by the Isola Group). In this manner, the substrate 3640 canmaintain the desired rigidity and dimensional integrity to providerepeatable thermal performance for each portion of the flow path 3620.

In some embodiments, the heater assembly 3630 and the substrate 3640(and any of the heater assemblies and substrates described herein) canbe a portion of a printed circuit board of a molecular diagnosticsdevice (e.g., any of the devices described herein, including the device6000 or the device 7000). Thus, the substrate 3640 can also supportand/or be coupled to electronic components that form the circuitry tocontrol the heater assembly 3630 as well as the overall diagnosticdevice (e.g., flow pumps, introduction of reagents, sample preparationoperations, or the like). For example, in some embodiments, thesubstrate 3640 (and any of the substrates or printed circuit boardlayers described herein) can be coupled to and/or support a processor, acontroller, or the like. In this manner, the heater assembly 3630 andthe substrate 3640 (and any of the heater assemblies and substratesdescribed herein) can be a portion of a printed circuit board thatperforms many different electronic functions, including controlling theamplification of the sample, controlling sample movement, and otherthermally-based functions described herein.

As shown, the substrate 3640 includes a first portion 3631, a secondportion 3632, and a third portion 3633. The third portion 3633 isbetween the first portion 3631 and the second portion 3632. Similarlystated, the third portion 3633 separates the first portion 3631 and thesecond portion 3632. The first heating element assembly 3650 is coupledto the first portion 3631 of the substrate 3640 and can produce a firstthermal output. The second heating element assembly 3660 is coupled tothe second portion 3632 of the substrate 3640 and can produce a secondthermal output. In some embodiments, the substrate 3640 can be coupledto the flow member 3610 such that the first portion 3631 of thesubstrate 3640 (and the first heating element assembly 3650) is alignedwith the first portion 3624 of the flow path 3620 and the second portion3632 of the substrate 3640 (and the second heating element assembly3660) is aligned with the second portion 3625 of the flow path 3620.This arrangement allows the first heating element assembly 3650 to heatthe first portion 3624 of the flow path 3620, and the second heatingelement assembly 3660 to heat the second portion 3625 of the flow path3620, as described herein.

The third portion 3633 of the substrate 3640 defines a series ofapertures 3641. Thus, the thermal conductivity (e.g., the thermalconductivity within the plane of the surface to which the heaters arecoupled) of the third portion 3633 of the substrate 3640 is impacted bythe shape, size and pattern of the apertures 3641. Because air has athermal conductivity of between about 0.01 W/m-K and 0.03 W/m-K and amaterial from which the substrate 3640 is fabricated (e.g., an FR-4grade printed circuit board material) can have a thermal conductivity ofbetween about 0.5 W/m-K and about 1.0 W/m-K, the inclusion of theapertures 3641 can result in the third portion 3633 of the substrate3640 having a thermal conductivity less than that of the first portion3631 of the substrate 3640 and the second portion 3632 of the substrate3640. In this manner, heat transfer within the substrate 3640 betweenthe first portion 3631 and the second portion 3632 is limited. Byminimizing the heat transfer between the first portion 3631 and thesecond portion 3632, accuracy of the heat flow from the first heatingelement 3650 and the second heating element 3660 to the flow member 3610can be improved.

The first heating element assembly 3650 is aligned with the firstportion 3624 of the flow path 3620, and thus can maintain the firstportion 3624 of the flow path 3620 at a first temperature. The firstheating element assembly 3650 can include one or more individual heatingelements (only one heating element is shown in FIG. 3). The secondheating element assembly 3660 is aligned with the second portion 3625 ofthe flow path 3620, and thus can maintain the second portion 3625 of theflow path 3620 at a second temperature. The second heating elementassembly 3660 can include one or more individual heating elements.Specifically, the second heating element assembly 3660 includes a firstelement 3664 and a second element 3665 that is electrically isolatedfrom the first element 3664. In this manner, an electrical current canbe conveyed to the first element 3664 independently from an electricalcurrent conveyed to the second element 3665. Similarly stated, thisarrangement allows for independent control of the first element 3664 andthe second element 3665.

As shown in FIG. 3, the elements of the second heating element assembly3660 are each aligned along the flow axis A_(F). Similarly stated, thesecond heating element assembly 3660 is segmented along the flow axisA_(F). This arrangement, along with the independent control of theelements of the second heating element assembly 3660 allow for accuratecontrol of heat flow to the portions of the flow path 3620. For example,in some embodiments, the first element 3664 of the second heatingelement assembly 3660 can be aligned with (and/or beneath) a firstchannel of the second portion 3625 of the flow path 3620. The secondelement 3665 of the second heating element assembly 3660 can be alignedwith (and/or beneath) a second (or last) channel of the second portion3625 of the flow path 3620. The segmented, independently controllabledesign allows the first element 3664 to produce a first thermal outputand the second element 3665 to produce a second thermal output that isdifferent from the first thermal output. By producing different thermaloutputs, the first channel and the second (or last) channel can be moreaccurately maintained at the desired temperature. For example, in someembodiments, the thermal design of the diagnostic device may result ingreater heat transfer away from the second (or last) channel of the flowpath 3620 (e.g., due to adjacent structures, internal air movement thatincreases convection transfer, or the like). In such situations, thesecond element 3665 can be controlled to provide a greater thermaloutput, thereby maintaining a consistent temperature between the firstchannel and the last channel. This, in turn, increases the overallaccuracy of the device.

Thus, the heater assembly 3630 and the flow member 3610 can establishmultiple temperature zones through which a sample can flow and/or becontained. Although the second heating element assembly 3660 is shown asincluding two independent heating elements, in other embodiments, eachof the first heating element assembly 3650 and the second heatingelement assembly 3660 can include any number of independent heatingelements (e.g. three elements, four elements, or more). Although thesecond heating element assembly 3660 is shown as being aligned with theflow axis A_(F), in other embodiments, each of the first heating elementassembly 3650 and the second heating element assembly 3660 can have anysuitable alignment. For example, in some embodiments, a heating elementassembly can be aligned with the flow channels. In some embodiments, aheating element assembly can include an element that extends beyond theflow path defined by a flow member (e.g., to minimize the effects ofheat “roll off” on an end channel of the flow path).

The heating elements can be any suitable heating element or collectionof heaters that can perform the functions described herein. For example,in some embodiments, any of the heating elements can be a single heatingelement that is thermally coupled to the flow member 3610, and that cancycle through multiple temperatures set points (e.g., between about 60 Cand about 90 C). Moreover, any of the heating elements can be of anysuitable design. For example, in some embodiments, any of the heatingelements can be a resistance heater, a thermoelectric device (e.g. aPeltier device), or the like. In some embodiments, any of the heatingelements can be resistance heaters that are lithographically produced onthe substrate 3640.

The flow member 3610 can be coupled to the heater assembly 3630 in anysuitable manner. For example, in some embodiments, the flow member 3610can be coupled to the heater assembly 3630 by a series of mechanicalfasteners, such as clamps, screws, or the like. In some suchembodiments, the fasteners can also function as heat sinks to allowaccurate control of the temperatures of the flow member 3610 and toavoid overheating. In other embodiments, the flow member 3610 can becoupled to the heater assembly 3630 by an adhesive (e.g., apressure-sensitive adhesive). Similarly stated, in some embodiments, theflow member 3610 can be chemically bonded to the heater assembly 3630.In yet other embodiments, the flow member 3610 can be coupled to theheater assembly 3630 by an adhesive (e.g., a pressure-sensitiveadhesive) and mechanical fasteners can be used to couple other structureand function as a heat sink. In this manner, the flow member 3610 isfixedly coupled to the heater assembly 3630. Said another way, in someembodiments, the flow member 3610 is not designed to be removed and/ordecoupled from the heater assembly 3630 during normal use (i.e., theflow member 3610 is irreversibly coupled to the heater assembly 3630and/or the substrate 3640). This arrangement facilitates a single-use,disposable device that includes the amplification module 3600.

In use, the first heating element assembly 3650 produces a first thermaloutput to maintain the first portion 3624 of the flow path 3620 (or thesample therein) at a first temperature. The first temperature can be,for example, between about 100 C and about 115 C (to heat the sample toabout 90 C; e.g., the “hot” temperature for a PCR thermal cycle). Thesecond heating element assembly 3660 produces a second thermal outputthat is different from the first thermal output, and that can also bedifferent between the first element 3664 and the second element 3665.The second thermal output can maintain the second portion 3625 of theflow path 3620 at a second temperature. The second temperature can be,for example, between about 60 C and about 75 C (to heat the sample toabout 60 C; e.g., the “cold” temperature for a PCR thermal cycle). Inthis manner, the heater assembly 3630 and the flow member 3610 canestablish multiple temperature zones within which a sample can flow orbe maintained. In other embodiments, the first temperature can bemaintained at a temperature suitable for cell lysis, and the secondtemperature can be maintained at a temperature suitable for anisothermal amplification operation. In this manner, the sample (and thenucleic acid(s) therein) can be prepared for amplification in the firstportion 3624 of the flow path 3620, and then amplified in the secondportion 3625 of the flow path 3620.

In some embodiments, the sample flowing within the flow path 3620 israpidly heated to about 90 C. To promote a rapid cooling down to about60 C, in some embodiments, heat must flow out of the sample (and thusthe flow member 3610). Thus, although the second temperature isdescribed as being hotter than the desired sample temperature, in otherembodiments, the output produced by the second heating element assembly3660 (or any of the heating elements described herein) can be such thatheat flows out of the flow path 3620 and/or the flow member 3610. Insuch embodiments, a current can still be supplied to the second heatingelement assembly 3660 to control the magnitude of the heat flow. In someembodiments, the second temperature can be, for example, between about40 C and about 45 C (to allow heat transfer away from the sample at acontrolled rate to facilitate maintaining the sample at about 60 C;e.g., the “cold” temperature for a PCR thermal cycle).

In some embodiments, the amplification module 3600 (and any of theamplification modules described herein) can perform any suitable type ofisothermal amplification process, including, for example, Loop MediatedIsothermal Amplification (LAMP), Nucleic Acid Sequence BasedAmplification (NASBA), which can be useful to detect target RNAmolecules, Strand Displacement Amplification (SDA), MultipleDisplacement Amplification (MDA), Ramification Amplification Method(RAM), or any other type of isothermal process.

In other embodiments, any suitable thermal reaction can be conductedeither of the first portion 3624 of the flow path 3620 or the secondportion 3625 of the flow path 3620. For example, in some embodiments,the temperature of the first portion 3624 (or any of the flow pathportions described herein) can be maintained to perform a hot-start onthe sample therein, while the temperature of the second portion 3625 canbe maintained or cycled to amplify the organisms therein. In otherembodiments, either of the first portion 3624 of the flow path 3620 orthe second portion 3625 of the flow path 3620 can be used to conduct alysing reaction, an inactivation reaction, and/or a detection reaction.

Although the substrate 1640 and the substrate 3640 are shown as definingone or more apertures (aperture 1641 and the apertures 3641,respectively), in some embodiments, an amplification module can includea substrate that does not define an aperture that separates heatersmounted thereto. For example, FIG. 4 is a schematic illustration of anamplification (or thermal reaction) module 4600, according to anembodiment. The amplification module 4600 is configured to perform athermal reaction (e.g., an amplification reaction) on an input of targetnucleic acid mixed with required reagents, and can be included in anysuitable diagnostic device. For example, the amplification module can beincluded in any of the diagnostic devices shown and described herein(including the device 6000 and the device 7000) or in InternationalPatent Publication No. WO2016/109691, entitled “Devices and Methods forMolecular Diagnostic Testing,” which is incorporated herein by referencein its entirety. In some embodiments, the amplification module 4600 isconfigured to conduct rapid amplification of an input target. In someembodiments, the amplification module 4600 is configured to generate anoutput copy number that reaches or exceeds the threshold of thesensitivity of an associated detection module.

The amplification module 4600 includes a flow member 4610 and a heaterassembly 4630. The flow member 4610 defines a flow path 4620 throughwhich a sample can flow from an inlet port 4621 to an outlet port 4622.The flow member 4610 defines a flow axis A_(F) that indicates theoverall direction of the flow through the flow member 4610. The flowpath 4620 is shaped and/or has a geometry such that various portions ofthe flow path 4620 (e.g., a first portion 4624 and a second portion4625) can be maintained at different temperatures by the heater assembly4630. In this manner, the amplification module 4600 can perform a “flowthrough” polymerase chain reaction (PCR) on the sample to amplify thetarget organism and/or portions of the DNA of the organism within thesample. Although the flow path 4620 is shown as being a serpentine path(or a path that includes multiple switchbacks to reverse the flow of thesample), in other embodiments, the flow path can have any suitable shapeand/or geometry.

The flow member 4610 (and any of the flow members described herein) canbe constructed from any suitable material and can have any suitabledimensions to facilitate the desired amplification performance for thedesired volume of sample. For example, in some embodiments, theamplification module 4600 (and any of the amplification modulesdescribed herein) can perform 1000× or greater amplification in a timeof less than 15 minutes. For example, in some embodiments, the flowmember 4610 (and any of the flow members described herein) isconstructed from at least one of a cyclic olefin copolymer or agraphite-based material. Such materials facilitate the desired heattransfer properties into the flow path 1620. Moreover, in someembodiments, the flow member 4610 (and any of the flow members describedherein) can have a thickness of less than about 0.5 mm. In someembodiments, the flow member 1610 (and any of the flow members describedherein) can have a volume about 150 microliters or greater, and the flowcan be such that at least 10 microliters of sample is amplified. Inother embodiments, at least 20 microliters of sample are amplified bythe methods and devices described herein. In other embodiments, at least30 microliters of sample are amplified by the methods and devicesdescribed herein. In yet other embodiments, at least 50 microliters ofsample are amplified by the methods and devices described herein.

The heater assembly 4630 includes a substrate 4640, a first heatingelement assembly 4650, and a second heating element assembly 4660, eachheating element assembly being coupled to the substrate 4640. Asdescribed herein, the first heating element assembly 4650 and the secondheating element assembly 4660 are each coupled between a first side 4657of the substrate 4640 and the flow member 4610. In this manner, theheater assembly 4630 is configured to maintain various portions of theflow path 4620 at different temperature set points to facilitate thedesired reaction (e.g., a thermal cycling amplification, an isothermamplification, a lysis reaction, or the like). The substrate 4640 can beany suitable substrate, such as for example, an electrically isolativesubstrate to which the first heating element assembly 4650 and secondheating element assembly 4660 are mounted. The substrate 4640 (and anyof the substrates described herein) can be constructed from any suitablematerial, such as, for example, a composite material including wovenglass and epoxy. In some embodiments, the substrate 4640 (and any of thesubstrates described herein) can be constructed from a material having aglass transition temperature (Tg) of greater than about 170 C. In otherembodiments, the substrate 4640 (and any of the substrates describedherein) can be constructed from a material having a glass transitiontemperature (Tg) of greater than about 180 C (e.g., material 370HRproduced by the Isola Group). In this manner, the substrate 4640 canmaintain the desired rigidity and dimensional integrity to providerepeatable thermal performance for each channel of the flow path 4620.

In some embodiments, the heater assembly 4630 and the substrate 4640(and any of the heater assemblies and substrates described herein) canbe a portion of a printed circuit board of a molecular diagnosticsdevice (e.g., any of the devices described herein, including the device6000 or the device 7000). Thus, the substrate 4640 can also supportand/or be coupled to electronic components that form the circuitry tocontrol the heater assembly 4630 as well as the overall diagnosticdevice (e.g., flow pumps, introduction of reagents, sample preparationoperations, or the like). For example, in some embodiments, thesubstrate 4640 (and any of the substrates or printed circuit boardlayers described herein) can be coupled to and/or support a processor, acontroller, or the like. In this manner, the heater assembly 4630 andthe substrate 4640 (and any of the heater assemblies and substratesdescribed herein) can be a portion of a printed circuit board thatperforms many different electronic functions, including controlling theamplification of the sample, controlling sample movement, and otherthermally-based functions described herein.

The first heating element assembly 4650 is aligned with the firstportion 4624 of the flow path 4620, and thus can maintain the firstportion 4624 of the flow path 4620 at a first temperature. The firsttemperature can be, for example, between about 100 C and about 115 C (toheat the sample to about 90 C; e.g., the “hot” temperature for a PCRthermal cycle). The first heating element assembly 4650 includes a firstelement 4661 and a second element 4662 that is electrically isolatedfrom the first element 4661. In this manner, an electrical current canbe conveyed to the first element 4661 independently from an electricalcurrent conveyed to the second element 4662. Similarly stated, thisarrangement allows for independent control of the first element 4661 andthe second element 4662.

The second heating element assembly 4660 is aligned with the secondportion 4625 of the flow path 4620, and thus can maintain the secondportion 4625 of the flow path 4620 at a second temperature. The secondtemperature can be, for example, between about 60 C and about 75 C (toheat the sample to about 60 C; e.g., the “cold” temperature for a PCRthermal cycle). The second heating element assembly 4660 includes afirst element 4664 and a second element 4665 that is electricallyisolated from the first element 4664. In this manner, an electricalcurrent can be conveyed to the first element 4664 independently from anelectrical current conveyed to the second element 4665. Similarlystated, this arrangement allows for independent control of the firstelement 4664 and the second element 4665. In some embodiments, thesample flowing within the flow path 4620 is rapidly heated to about 90C. To promote a rapid cooling down to about 60 C, in some embodiments,heat must flow out of the sample (and thus the flow member 4610). Thus,although the second temperature is described as being hotter than thedesired sample temperature, in other embodiments, the output produced bythe second heating element assembly 4660 (or any of the heating elementsdescribed herein) can be such that heat flows out of the flow path 4620and/or the flow member 4610. In such embodiments, a current can still besupplied to the second heating element assembly 4660 to control themagnitude of the heat flow. In some embodiments, the second temperaturecan be, for example, between about 40 C and about 45 C (to allow heattransfer away from the sample at a controlled rate to facilitatemaintaining the sample at about 60 C; e.g., the “cold” temperature for aPCR thermal cycle).

As shown in FIG. 4, the elements of the first heating element assembly4650 and the elements of the second heating element assembly 4660 areeach aligned along the flow axis A_(F). Similarly stated, the firstheating element assembly 4650 and the second heating element assembly4660 are segmented along the flow axis A_(F). This arrangement, alongwith the independent control of the elements of the first heatingelement assembly 4650 and the second heating element assembly 4660 allowfor accurate control of heat flow to the portions of the flow path 4620.For example, in some embodiments, the first element 4661 of the firstheating element assembly 4650 can be aligned with (and/or beneath) afirst channel of the flow path 4620 (e.g., along the first, or “hot,”portion of the flow member 4610). The second element 4662 of the firstheating element assembly 4650 can be aligned with (and/or beneath) asecond (or last) channel of the flow path 4620 (e.g., also along thefirst, or “hot,” portion of the flow member 4610). The segmented,independently controllable design allows the first element 4661 toproduce a first thermal output and the second element 4662 to produce asecond thermal output that is different from the first thermal output.By producing different thermal outputs, the hot portion of the firstchannel and the hot portion of the second (or last) channel can be moreaccurately maintained at the desired temperature. For example, in someembodiments, the thermal design of the diagnostic device may result ingreater heat transfer away from the second (or last) channel of the flowpath 4620 (e.g., due to adjacent structures, internal air movement thatincreases convection transfer, or the like). In such situations, thesecond element 4662 can be controlled to provide a greater thermaloutput, thereby maintaining a consistent temperature between the firstchannel and the last channel. This, in turn, increases the overallaccuracy of the device.

Similarly, in some embodiments, the first element 4664 of the secondheating element assembly 4660 can be aligned with (and/or beneath) afirst channel of the flow path 4620 (e.g., along the second, or “cold,”portion of the flow member 4610). The second element 4665 of the secondheating element assembly 4660 can be aligned with (and/or beneath) asecond (or last) channel of the flow path 4620 (e.g., also along thesecond, or “cold,” portion of the flow member 4610). The segmented,independently controllable design allows the first element 4664 toproduce a first thermal output and the second element 4665 to produce asecond thermal output that is different from the first thermal output.By producing different thermal outputs, the cold portion of the firstchannel and the cold portion of the second (or last) channel can be moreaccurately maintained at the desired temperature. For example, in someembodiments, the thermal design of the diagnostic device may result ingreater heat transfer away from the second (or last) channel of the flowpath 4620 (e.g., due to adjacent structures, internal air movement thatincreases convection transfer, or the like). In such situations, thesecond element 4665 can be controlled to provide a greater thermaloutput, thereby maintaining a consistent temperature between the firstchannel and the last channel. This, in turn, increases the overallaccuracy of the device.

Thus, the heater assembly 4630 and the flow member 4610 can establishmultiple temperature zones through which a sample can flow, and candefine a desired number of amplification cycles to ensure the desiredtest sensitivity (e.g., at least 30 cycles, at least 34 cycles, at least36 cycles, at least 38 cycles, or at least 40 cycles). Although each ofthe first heating element assembly 4650 and the second heating elementassembly 4660 are shown as including two independent heating elements,in other embodiments, each of the first heating element assembly 4650and the second heating element assembly 4660 can include any number ofindependent heating elements (e.g. three elements, four elements, ormore). Although each of the first heating element assembly 4650 and thesecond heating element assembly 4660 are shown as being aligned with theflow axis A_(F), in other embodiments, each of the first heating elementassembly 4650 and the second heating element assembly 4660 can have anysuitable alignment. For example, in some embodiments, a heating elementassembly can be aligned with the flow channels. In some embodiments, aheating element assembly can include an element that extends beyond theflow path defined by a flow member (e.g., to minimize the effects ofheat “roll off” on an end channel of the flow path).

The heating elements 4661, 4662, 4664, 4665 can be any suitable heatingelement or collection of heaters that can perform the functionsdescribed herein. For example, in some embodiments, any of the heatingelements can be a single heating element that is thermally coupled tothe flow member 4610, and that can cycle through multiple temperaturesset points (e.g., between about 60 C and about 90 C). Moreover, any ofthe heating elements can be of any suitable design. For example, in someembodiments, any of the heating elements can be a resistance heater, athermoelectric device (e.g. a Peltier device), or the like. In someembodiments, any of the heating elements can be resistance heaters thatare lithographically produced on the substrate 4640.

The flow member 4610 can be coupled to the heater assembly 4630 in anysuitable manner. For example, in some embodiments, the flow member 4610can be coupled to the heater assembly 4630 by a series of mechanicalfasteners, such as clamps, screws, or the like. In some suchembodiments, the fasteners can also function as heat sinks to allowaccurate control of the temperatures of the flow member 4610 and toavoid overheating. In other embodiments, the flow member 4610 can becoupled to the heater assembly 4630 by an adhesive (e.g., apressure-sensitive adhesive). Similarly stated, in some embodiments, theflow member 4610 can be chemically bonded to the heater assembly 4630.In this manner, the flow member 4610 is fixedly coupled to the heaterassembly 4630. Said another way, in some embodiments, the flow member4610 is not designed to be removed and/or decoupled from the heaterassembly 4630 during normal use. This arrangement facilitates asingle-use, disposable device that includes the PCR module 4600.

Although the first heating element assembly 4650 and the second heatingelement assembly 4660 are shown and described as being coupled to thefirst side 4657 of the substrate 4640, in other embodiments, the firstheating element assembly 4650 can be coupled to a first (e.g., front)side of the substrate 4640 and the second heating element assembly 4660can be coupled to a second (e.g., back) side of the substrate 4640. Insuch embodiments, the flow member can be configured to wrap around thesubstrate. In yet other embodiments, a heating element can be disposedwithin a substrate (e.g., on an inner layer of a multi-layerconstruction).

In some embodiments, a heater assembly of an amplification (or thermalreaction) module can be fabricated lithographically and/or can beintegral with a printed circuit board. For example, FIG. 5 shows anexploded view of an amplification module 5600, according to anembodiment that includes a multi-layer heater assembly 5630 fabricatedusing lithography. The amplification module 5600 is configured toperform a thermal reaction (e.g., an amplification reaction) on an inputof target nucleic acid mixed with required reagents, and can be includedin any suitable diagnostic device. For example, the amplification modulecan be included in any of the diagnostic devices shown and describedherein (including the device 6000 and the device 7000) or inInternational Patent Publication No. WO2016/109691, entitled “Devicesand Methods for Molecular Diagnostic Testing,” which is incorporatedherein by reference in its entirety. In some embodiments, theamplification module 5600 is configured to conduct rapid amplificationof an input target. In some embodiments, the amplification module 5600is configured to generate an output copy number that reaches or exceedsthe threshold of the sensitivity of an associated detection module(e.g., the detection module 3800 described below).

As described below, the heater assembly 5630 is a portion of a printedcircuit board of a molecular diagnostics device (e.g., any of thedevices described herein, including the device 6000 and the device7000). Thus, the substrate, the circuit board layers and/or otherstructure of the heater assembly 5630 also support and/or is coupled toelectronic components (not shown in FIGS. 5-17) that form the circuitryto control the heater assembly 5630 as well as the overall diagnosticdevice (e.g., flow pumps, introduction of reagents, sample preparationoperations, or the like). For example, in some embodiments, a portion ofthe heater assembly 5630 (and any of the substrates or printed circuitboard layers described herein) can be coupled to and/or support aprocessor, a controller, or the like. In this manner, the heaterassembly 5630 can be a portion of a printed circuit board that performsmany different electronic functions, including controlling theamplification of the sample, controlling sample movement, and otherthermally-based functions described herein.

Referring to FIG. 5, the amplification module 5600 includes a flowmember 5610, a circuit board (or heater) assembly 5630, and a heat sink5690. The flow member 5610 is coupled between the circuit board assembly5630 and the heat sink 5690. As shown in FIGS. 5-7, the flow member 5610includes a body 5617 and a lid 5619. The body 5617 is covered with athin plastic lid 5619 which is attached with a pressure sensitiveadhesive 5618. The lid 5619 allows for easy flow of thermal energy fromthe circuit board assembly 5630. In some embodiments, the flow member5610 also contains features to allow other parts of the assembly (e.g.,the circuit board assembly 5630) to align with the features on the flowmember 5610, as well as features to allow the fluidic connections to bebonded correctly. The adhesive 5618 used to attach the lid 5619 isselected to be “PCR-safe” and is formulated to not deplete the reagentor target organism concentrations in the PCR reaction.

The body 5617 defines a flow path 5620 through which a sample can flowfrom an inlet port 5621 to an outlet port 5622. The flow member 5620defines a flow axis A_(F) that indicates the overall direction of theflow through the flow member 5610. As shown, the amplification flow pathhas a curved, switchback or serpentine pattern. More specifically, theflow member (or chip) 5610 has two serpentine patterns—an amplificationpattern and a hot-start pattern 5623. The amplification pattern allowsfor amplification (i.e., PCR in this instance) to occur while thehot-start pattern 5623 accommodates the hot-start conditions of the PCRenzyme.

The serpentine arrangement provides a high flow length while maintainingthe overall size of the amplification module 5600 within the desiredlimits. Moreover, the serpentine shape allows the flow path 5620 tointersect circuit board assembly 5630 at multiple locations (e.g., alongthe flow axis A_(F)). This arrangement can produce distinct “heatingzones” throughout the flow path 5620, such that the amplification module5600 can perform a “flow through” PCR when the sample flows throughmultiple different temperature regions. As shown, the flow path 5620 isshaped and/or has a geometry such that various portions of the flow path5620 (e.g., a first portion 5624, a second portion 5625, and a thirdportion 5626) can be maintained at different temperatures by the circuitboard assembly 5630. Specifically, as shown in FIG. 7, the circuit boardassembly 5630 is coupled to the flow member 5610 to establish threetemperature zones identified by the dashed lines: a first (i.e., centralor “hot”) temperature zone 5611, a second (or end) temperature zone5612, and a third (or end) temperature zone 5613. The circuit boardassembly 5630 and the flow member 5610 also establish a fourth (or hotstart) temperature zone 5614. In use, the second temperature zone 5612(which includes the second portion 5625 of the flow path 5620) and thethird temperature zone 5613 (which includes the third portion 5626 ofthe flow path 5620) can be maintained at a temperature of about 60degrees Celsius (and/or at a surface temperatures such that the fluidflowing therethrough reaches a temperature of about 60 degrees Celsius).The first temperature zone 5611 (which includes the first portion 5624of the flow path 5620) can be maintained at a temperature of about 90degrees Celsius (and/or at a surface temperatures such that the fluidflowing therethrough reaches a temperature of about 90 degrees Celsius).Thus, in use the first portion 5624, the second portion 5625, and thethird portion 5626 can be maintained at two different temperatures bythe circuit board assembly 5630.

As shown, the serpentine pattern establishes 40 different zones of“cold-to-hot-to-cold;” or 40 amplification cycles. In other embodiments,however, the flow member 5610 (or any of the other flow membersdescribed herein) can define any suitable number of switchbacks oramplification cycles to ensure the desired test sensitivity. In someembodiments, the flow member can define at least 30 cycles, at least 34cycles, at least 36 cycles, at least 38 cycles, or at least 40 cycles.

The dimensions of the flow channel 5620 in the flow member 5610 impactthe temperature conditions of the PCR and dictate the overall dimensionsof the chip, and thus affect the overall power consumption of theamplification module 5600. For example, a deeper, narrower channel willdevelop a larger gradient in temperature from the side closest to thelid 5619 to the bottom (resulting in lower PCR efficiency). Thisarrangement, however, requires less overall space since the channelswill take up less overall surface area facing the heater assembly 5630(and thus require less energy to heat). The opposite holds true for awide and shallow channel. In some embodiments, the depth of the flowchannel 5620 is about 0.15 mm and the width of the flow channel 5620 isbetween about 1.1 mm and about 1.3 mm. More particularly, in someembodiments, the flow channel 5620 has a width of about 1.1 mm in the“narrow” sections (that are within the second temperature zone 5612 andthe third temperature zone 5613) and about 1.3 mm in the “wide” section(that falls within the first temperature zone 5611). In someembodiments, the overall path length is about 960 mm (including both theamplification portion and the hot start portion 5623). In suchembodiments, the total path length of the amplification portion is about900 mm. This produces a total volume of the flow channel 5620 of about160 μl (including the hot start portion 5623) and about 150 μl (withoutthe hot start portion 5623). In some embodiments, the separation betweeneach parallel path is between about 0.4 mm and about 0.6 mm.

The flow member 5610 and/or body 5617 can be constructed from anysuitable material, and can have any suitable thickness. For example, insome embodiments, the flow member 5610 and/or body 5617 (and any of theflow members described herein) can be molded from COC (Cyclic OlefinCopolymer) plastic, which has inherent barrier properties and lowchemical interactivity. In other embodiments, the flow member 5610and/or body 5617 (and any of the flow members described herein) can beconstructed from a graphite-based material (for improved thermalproperties). The overall thickness of the flow member 5610 can be lessthan about 0.5 mm, less than about 0.4 mm, less than about 0.3 mm orless than about 0.2 mm.

The flow member 5610 can be coupled to the circuit board assembly 5630in any suitable manner. For example, in some embodiments, the flowmember 5610 can be coupled to the heater assembly 5630 at least in partby the mechanical fasteners used to couple the heat sink 5690 to thecircuit board assembly 5630. In some such embodiments, the fasteners canalso function as heat sinks to allow accurate control of thetemperatures of the flow member 5610 and to avoid overheating. In otherembodiments, the flow member 5610 can be coupled to the heater assembly5630 by an adhesive (e.g., a pressure-sensitive adhesive). Similarlystated, in some embodiments, the flow member 5610 can be chemicallybonded to the heater assembly 5630. In this manner, the flow member 5610is fixedly and irreversibly coupled to the heater assembly 5630. Saidanother way, in some embodiments, the flow member 5610 is not designedto be removed and/or decoupled from the heater assembly 5630 duringnormal use. This arrangement facilitates a single-use, disposable devicethat includes the PCR module 5600.

The circuit board (or heater) assembly 5630 is a multi-layer circuitboard having a first side 5637 and a second side 5638. FIGS. 9 and 10show an exploded view of each layer of the circuit board assembly 5630.FIG. 9 shows various layers of the circuit board 5630, including thecopper traces fabricated thereon, and FIG. 10 shows a structuralrepresentation of each layer. Although FIG. 9 shows the four layersincluding copper (or a conductive portion), in some embodiments, a thinfilm substrate (not shown) separates the adjacent copper layers. Asshown, the circuit board assembly 5630 includes a substrate 5640 havinga first side 5657 and a second side 5658, a first (or outer heater)layer 5646, a second (or inner heater) layer 5647, a third (or innersensor) layer 5648, and a fourth (or outer sensor) layer 5649. Thesubstrate 5640 provides structural support, and is constructed from anelectrically isolative material upon which the four layers arefabricated using lithographic procedures. The substrate 5640 (and any ofthe substrates described herein) can be constructed from any suitablematerial, such as, for example, a composite material including wovenglass and epoxy. In some embodiments, the substrate 5640 (and any of thesubstrates described herein) can be constructed from a material having aglass transition temperature (Tg) of greater than about 170 C. In otherembodiments, the substrate 5640 (and any of the substrates describedherein) can be constructed from a material having a glass transitiontemperature (Tg) of greater than about 180 C (e.g., material 370HRproduced by the Isola Group). In this manner, the substrate 5640 canmaintain the desired rigidity and dimensional integrity to providerepeatable thermal performance for each channel of the flow path 5620.

FIGS. 11-14 show top views of each of the four layers of the printedcircuit board assembly 5630. Specifically, FIG. 11 shows a top view ofthe first (or outer heater) layer 5646. FIG. 12 shows a top view of thesecond (or inner heater) layer 5647. FIG. 13 shows a top view of thethird layer 5648. FIG. 14 shows a top view of the fourth (or outersensor) layer 5649. As discussed below, the fourth layer 5649 alsoincludes the fifth heater assembly 5669. Each of these layers isdiscussed below within the description of the overall printed circuitboard assembly 5630.

Referring again to FIG. 5, the circuit board assembly 5630 defines aseries of apertures (also referred to as openings, cut-outs, or vias)that separate the circuit board assembly 5630 into several differentportions (or heating zones). Specifically, the circuit board assembly5630 defines a first set of apertures 5641 that separates a firstportion (or heating zone) 5631 of the assembly 5630 from a secondportion (or heating zone) 5632 of the assembly 5630. The first set ofapertures 5641 includes three openings that are elongated along the flowaxis A_(F), and that are separated by two connection lugs 5651 (only oneof the connection lugs 5651 is identified). Thus, the first set ofapertures 5641 produces a longitudinally oriented thermal barrierbetween the first heating zone 5631 and the second heating zone 5632.Similarly, the circuit board assembly 5630 defines a second set ofapertures 5642 that separates the first portion (or heating zone) 5631of the assembly 5630 from a third portion 5633 (or heating zone) of theassembly 5630. The second set of apertures 5642 includes three openingsthat are elongated along the flow axis A_(F), and that are separated bytwo connection lugs 5652 (only one of the connection lugs 5652 isidentified). Thus, the second set of apertures 5642 produces alongitudinally oriented thermal barrier between the first heating zone5631 and the third heating zone 5633.

The first heating zone 5631 is disposed between the second heating zone5632 and the third heating zone 5633. Moreover, when the circuit boardassembly 5630 is coupled to the flow member 5610, the first portion 5631is aligned with the first (or “hot”) temperature zone 5611, the secondportion 5632 is aligned with the second (or “cold”) temperature zone5612, and the third portion 5633 is aligned with the third (or “cold”)temperature zone 5613. This is illustrated in the cross-sectional viewshown in FIG. 15, which shows the first temperature zone 5611 of theflow member (identified by the dashed lines) being surrounded and/orisolated by the first set of apertures 5641 and the second set ofapertures 5642. This arrangement allows the first heater assembly 5650located within first heating zone 5631 to heat the first temperaturezone 5611 of the flow member 5610. In some embodiments, the first heaterassembly 5650 can be controlled to maintain the first temperature zone5611 at a temperature of about 90 degrees Celsius (and/or at a surfacetemperatures such that the fluid flowing therethrough reaches atemperature of about 90 degrees Celsius). This arrangement furtherallows the second heater assembly 5660 located within second heatingzone 5632 to heat the second temperature zone 5612 of the flow member5610. In some embodiments, the second heater assembly 5660 can becontrolled to maintain the second temperature zone 5612 at a temperatureof about 60 degrees Celsius (and/or at a surface temperatures such thatthe fluid flowing therethrough reaches a temperature of about 60 degreesCelsius). This arrangement further allows the third heater assembly 5670located within third heating zone 5633 to heat the third temperaturezone 5613 of the flow member 5610. In some embodiments, the third heaterassembly 5670 can be controlled to maintain the third temperature zone5613 at a temperature of about 60 degrees Celsius (and/or at a surfacetemperatures such that the fluid flowing therethrough reaches atemperature of about 60 degrees Celsius). In this manner, the heaterassembly 5630 and the flow member 5610 can establish multipletemperature zones through which a sample can flow, and can define adesired number of amplification cycles to ensure the desired testsensitivity (e.g., at least 30 cycles, at least 34 cycles, at least 36cycles, at least 38 cycles, or at least 40 cycles).

Referring to FIG. 11, the first heating zone 5631 of the printed circuitboard assembly 5630 includes a first heater assembly 5650. The firstheater assembly 5650 includes a first heating element 5661, a secondheating element 5662, and a third heating element 5663, each of which iselectrically isolated from the other two heating elements in the firstheater assembly 5650. Said another way, each of the first heatingelement 5661, the second heating element 5662, and the third heatingelement 5663 is separate from (or electrically isolated from) theothers. In this manner, an electrical current can be conveyed to each ofthe first heating element 5661, the second heating element 5662, and thethird heating element 5663 independently from an electrical currentbeing conveyed to the other heating elements of the first heaterassembly 5650. This arrangement allows for independent control of thefirst heating element 5661, the second heating element 5662, and thethird heating element 5663. Each of the first heating element 5661, thesecond heating element 5662, and the third heating element 5663 areconductive traces that are fabricated on the first layer 5646 bylithographic techniques. Although the first heater assembly 5650 isshown as being fabricated in the first layer 5646, in other embodiments,the first heater assembly 5650 can be fabricated in any layer of thecircuit board assembly 5630.

The second heating zone 5632 of the printed circuit board assembly 5630includes a second heater assembly 5660. The second heater assembly 5660includes a first heating element 5664 and a second heating element 5665,each being electrically isolated from the other. Said another way, thefirst heating element 5664 is separate from the second heating element5665, and vice-versa. In this manner, an electrical current can beconveyed to the first heating element 5664 independently from anelectrical current being conveyed to the second heating element 5665,and vice-versa. This arrangement allows for independent control of thefirst heating element 5664 and the second heating element 5665. Thefirst heating element 5664 and the second heating element 5665 are eachconductive traces that are fabricated on the first layer 5646 bylithographic techniques. Although the second heater assembly 5660 isshown as being fabricated in the first layer 5646, in other embodiments,the second heater assembly 5660 can be fabricated in any layer of thecircuit board assembly 5630.

The third heating zone 5633 of the printed circuit board assembly 5630includes a third heater assembly 5670. The third heater assembly 5670includes a first heating element 5666 and a second heating element 5667,each being electrically isolated from the other. Said another way, thefirst heating element 5666 is separate from the second heating element5667, and vice-versa. In this manner, an electrical current can beconveyed to the first heating element 5666 independently from anelectrical current being conveyed to the second heating element 5667,and vice-versa. This arrangement allows for independent control of thefirst heating element 5666 and the second heating element 5667. Thefirst heating element 5666 and the second heating element 5667 are eachconductive traces that are fabricated on the first layer 5646 bylithographic techniques. Although the third heater assembly 5670 isshown as being fabricated in the first layer 5646, in other embodiments,the third heater assembly 5670 can be fabricated in any layer of thecircuit board assembly 5630.

In use, the first heater assembly 5650 produces a thermal output tomaintain the first temperature zone 5611 of the flow member 5610 at afirst temperature. The first temperature can be, for example, betweenabout 100 C and 115 C (to heat the sample therein to about 90 C; e.g.,the “hot” temperature for a PCR thermal cycle). Additionally, thesegmented, independently controllable design allows the first heatingelement 5661 to produce a first thermal output, the second heatingelement 5662 to produce a second thermal output, and the third heatingelement 5663 to produce a third thermal output, each of which can bedifferent from the others. By producing different thermal outputs, thehot portion of the flow channel 5620 can be more accurately maintainedat the desired temperature. For example, in some embodiments, thethermal design of the diagnostic device may result in greater heattransfer away from the first or last channels of the flow path 5620(e.g., due to adjacent structures, internal air movement that increasesconvection transfer, or the like). In such situations, the first heatingelement 5661 and the third heating element 5663 can be controlled toprovide a greater thermal output, thereby maintaining a consistenttemperature between the first channel and the last channel. This, inturn, increases the overall accuracy of the device.

The second heater assembly 5660 produces a thermal output to maintainthe second temperature zone 5612 of the flow member 5610 at a secondtemperature. The second temperature can be different from the firsttemperature, and can be, for example, about 60 C to about 75 C (to heatthe sample therein to about 60 C; e.g., the “cold” temperature for a PCRthermal cycle). Additionally, the segmented, independently controllabledesign allows the first heating element 5664 to produce a first thermaloutput and the second heating element 5665 to produce a second thermaloutput different from the first thermal output. By producing differentthermal outputs, the cold portion of the flow channel 5620 can be moreaccurately maintained at the desired temperature. For example, in someembodiments, the thermal design of the diagnostic device may result ingreater heat transfer away from the first or last channels of the flowpath 5620 (e.g., due to adjacent structures, internal air movement thatincreases convection transfer, or the like). In such situations, thefirst heating element 5664 and the third heating element 5665 can becontrolled to provide different thermal outputs, thereby maintaining aconsistent temperature between the first channel and the last channel.This, in turn, increases the overall accuracy of the device.

In some embodiments, the sample flowing within the flow path 5620 israpidly heated to about 90 C. To promote a rapid cooling down to about60 C, in some embodiments, heat must flow out of the sample (and thusthe flow member 5610). Thus, although the second temperature isdescribed as being hotter than the desired sample temperature, in otherembodiments, the output produced by the second heater assembly 5660 (orany of the heating elements described herein) can be such that heatflows out of the flow path 5620 and/or the flow member 5610. In suchembodiments, a current can still be supplied to the second heaterassembly 5660 to control the magnitude of the heat flow. In someembodiments, the second temperature can be, for example, between about40 C and about 45 C (to allow heat transfer away from the sample at acontrolled rate to facilitate maintaining the sample at about 60 C;e.g., the “cold” temperature for a PCR thermal cycle).

The third heater assembly 5670 produces a thermal output to maintain thethird temperature zone 5613 of the flow member 5610 at a thirdtemperature. The third temperature can be different from the firsttemperature and/or the second temperature. In some embodiments, thethird temperature can be the same as the second temperature, and can be,for example, about 60 C to about 75 C (to heat the sample therein toabout 60 C; e.g., the “cold” temperature for a PCR thermal cycle).Additionally, the segmented, independently controllable design allowsthe first heating element 5666 to produce a first thermal output and thesecond heating element 5667 to produce a second thermal output differentfrom the first thermal output. By producing different thermal outputs,the cold portion of the flow channel 5620 can be more accuratelymaintained at the desired temperature. For example, in some embodiments,the thermal design of the diagnostic device may result in greater heattransfer away from the first or last channels of the flow path 5620(e.g., due to adjacent structures, internal air movement that increasesconvection transfer, or the like). In such situations, the first heatingelement 5665 and the third heating element 5667 can be controlled toprovide different thermal outputs, thereby maintaining a consistenttemperature between the first channel and the last channel. This, inturn, increases the overall accuracy of the device.

As described above, the first set of apertures 5641 produces alongitudinally oriented thermal barrier between the first heating zone5631 and the second heating zone 5632, and the second set of apertures5642 produces a longitudinally oriented thermal barrier between thefirst heating zone 5631 and the third heating zone 5633. Thus, the firstset of apertures 5641 and the second set of apertures 5642 collectivelythermally isolate the first heating zone 5631 of the circuit boardassembly 5630. By minimizing the heat transfer between the first heatingzone 5631, the second heating zone 5632, and the third heating zone5633, accuracy and control of the temperature to which each heating zoneis heated can be improved.

Referring to FIG. 11, the connection lugs (or portions) 5651 thatseparate the first set of apertures 5641 into three openings are offsetfrom the connection lugs (or portions) 5652 that separate the second setof apertures 5642 into three openings. Similarly stated, a connectionlug 5651 is located at a different longitudinal position than acorresponding connection lug 5652. Said another way, the connection lugs5651 are positioned at a different location along the flow axis A_(F)than are the connection lugs 5652. This arrangement allows each of theconnection lugs 5651 and the connection lugs 5652 to be positioned below(or aligned with) a different channel of the flow path 5620 when thecircuit board assembly 5630 is coupled to the flow member 5610. As anexample, this arrangement allows a connection lug 5651 to be alignedwith, for example, the tenth channel within the flow path 5620 while thecorresponding connection lug 5652 is aligned with, for example, thetwelfth channel within the flow path 5620. Because the thermalperformance of the first heating zone 5631 in the areas adjacent theconnection lugs 5651 and 5652 is different than the thermal performanceat other spatial locations, the offset arrangement of the connectionlugs minimizes any differences in the temperature of the flow channel.This, in turn, increases the overall accuracy of the device.

The connection lugs (or portions) 5651 and the connection lugs (orportions) 5652 can be of any suitable size. For example, the width ofthe connection lugs 5651 and the connection lugs 5652 can be such thatduring normal use deflection and/or warping of the circuit boardassembly 5630 is minimized. Any such warping could result in changes inthe contact between the flow member 5610 and the heater assemblies,thereby resulting in variation in the temperatures maintained within theflow channel 5620. In some embodiments, the width of the connection lugs5651 and/or the connection lugs 5652 can be about 5 percent of the widthof one of the openings from the first set of apertures 5641 and/or thesecond set of apertures 5642. In other embodiments, the width of theconnection lugs 5651 and/or the connection lugs 5652 can be betweenabout 5 percent and about 10 percent of the width of one of the openingsfrom the first set of apertures 5641 and/or the second set of apertures5642. In other embodiments, the width of the connection lugs 5651 and/orthe connection lugs 5652 can be between about 10 percent and about 20percent of the width of one of the openings from the first set ofapertures 5641 and/or the second set of apertures 5642.

In addition to including three heating zones for the PCR reaction, thecircuit board assembly 5630 also defines a third aperture 5643 thatseparates a fourth portion (or heating zone) 5634 from the other heatingzones. The third aperture 5643 is elongated substantially perpendicularto the flow axis A_(F), and thus produces a laterally-oriented thermalbarrier between the fourth heating zone 5634 and the amplificationheating zones (i.e., the first heating zone 5631, the second heatingzone 5632, and the third heating zone 5633). Although shown as being asingle opening, in other embodiments the fourth heating zone 5634 can beseparated by a series of apertures and connection lugs.

As shown, the fourth heating zone 5634 is disposed at an end portion ofthe circuit board assembly 5630, opposite the first heating zone, 5631,the second heating zone 5632, and the third heating zone 5633. Moreover,when the circuit board assembly 5630 is coupled to the flow member 5610,the fourth portion 5634 is aligned with the hot-start pattern 5623 ofthe flow member 5610. This arrangement allows the fourth heater assembly5668 located within fourth heating zone 5634 to heat the hot-startpattern 5623 of the flow member 5610. The hot-start portion 5623 reducesnon-specific amplification and allows the use of certain PCR reagentsthat remain inactive until heated. In some embodiments, the first heaterassembly 5650 can be controlled to maintain the first temperature zone5611 at a temperature of between about 45 degrees Celsius and about 95degrees Celsius (and/or at a surface temperatures such that the fluidflowing therethrough reaches a temperature between about 45 degreesCelsius and about 95 degrees Celsius).

The fourth heating zone 5634 of the printed circuit board assembly 5630includes a fourth heater assembly 5668. The fourth heater assembly 5668includes a single heating element that is electrically isolated from theheating elements included in the other heating assemblies (e.g., thefirst heating assembly 5650). In this manner, an electrical current canbe conveyed to the fourth heater assembly 5668 independently from anelectrical current being conveyed to the other heating elements of thefirst heater assembly 5650, the second heater assembly 5660 and/or thethird heater assembly 5670. This arrangement allows for independentcontrol of the hot-start portion of the amplification module 5600. Theheating element of the fourth heater assembly 5668 includes conductivetraces that are fabricated on the first layer 5646 by lithographictechniques. Although the fourth heater assembly 5668 is shown as beingfabricated in the first layer 5646, in other embodiments, the fourthheater assembly 5668 can be fabricated in any layer of the circuit boardassembly 5630. Moreover, although the fourth heater assembly 5668 isshown as including a single heating element, in other embodiments, thefourth heater assembly 5668 can include any number if independentlycontrollable (or segmented) heating elements.

The circuit board assembly 5630 defines a fourth set of apertures 5644that separates the fifth portion (or heating zone) 5635 from the otherportions of the board. The fourth set of apertures 5644 includes threeopenings that are separated by two connection lugs (the connection lugsare not identified). The fourth set of apertures 5644 includes oneopening that is elongated along the flow axis A_(F), and two openingsthat are elongated substantially perpendicular to the flow axis A_(F).Thus, the fourth set of apertures 5644 produces thermal barrier thatsurrounds the fifth heating zone 5635.

As shown, the fifth heating zone 5635 is disposed at a side portion ofthe circuit board assembly 5630, opposite the first heating zone, 5631,the second heating zone 5632, and the third heating zone 5633. Moreover,when the circuit board assembly 5630 is coupled to the flow member 5610,the fifth heating zone 5635 is spaced apart from the flow member 5610.Thus, the heat produced by fifth heater assembly 5669 (see FIG. 14) isnot directed towards any portion of the flow path 5620. Rather, as shownin FIG. 16, the fifth heating zone 5635 is aligned with a detectionmodule 5800. This arrangement allows the fifth heater assembly 5669located within fifth heating zone 5635 to heat portions of the detectionmodule 5800 to facilitate post-amplification detection of a targetorganism. In this manner, the circuit board assembly 5630 can functionto heat both the flow member 5610 to facilitate amplification and thedetection module 5800 to facilitate detection.

Referring to FIG. 14, the fifth heating zone 5635 of the printed circuitboard assembly 5630 includes a fifth heater assembly 5669. The fifthheater assembly 5669 includes a single heating element that iselectrically isolated from the heating elements included in the otherheating assemblies (e.g., the first heating assembly 5650). In thismanner, an electrical current can be conveyed to the fifth heaterassembly 5669 independently from an electrical current being conveyed tothe other heating elements of the first heater assembly 5650, the secondheater assembly 5660, the third heater assembly 5670, and/or the fourthheater assembly 5668. This arrangement allows for independent control ofthe detection module 5800. The heating element of the fifth heaterassembly 5669 includes conductive traces that are fabricated on thefourth layer 5646 by lithographic techniques. Although the fifth heaterassembly 5669 is shown as being fabricated in the fourth layer 5649, inother embodiments, the fifth heater assembly 5669 can be fabricated inany layer of the circuit board assembly 5630. Moreover, although thefifth heater assembly 5669 is shown as including a single heatingelement, in other embodiments, the fifth heater assembly 5669 caninclude any number if independently controllable (or segmented) heatingelements.

Referring to FIGS. 16 and 17, the detection module 5800 is coupled tothe second side 5638 of the printed circuit board assembly 5630.Similarly stated, the detection module 5800 is coupled to the oppositeside of the printed circuit board assembly 5630 than the flow member5610. The detection module 5800 is configured to receive output from theamplification module 5600 (i.e., from the flow member 5610) and reagentsfrom a reagent module (not shown) to produce an output to indicatepresence or absence of target organism in the initial input sample. Thedetection module 5800 can also produce an output to indicate the generalcorrect operation of the test (positive control and negative control).For example, in some embodiments, the detection module 5800 can produceone or more colorimetric outputs. The detection module 6800 can be anyof the detection modules shown and described in International PatentPublication No. WO2016/109691, entitled “Devices and Methods forMolecular Diagnostic Testing,” which is incorporated herein by referencein its entirety.

The detection module 5800 includes a detection flow cell that defines adetection chamber/channel 5812 having a series of inlet and outletportions, through which the sample and the reagents are conveyed toproduce the detected output. The detection chamber 5812 includes a “readlane” that includes one or more detection surfaces or zones. In someembodiments, the detection surfaces can be chemically modified tocontain hybridization probes (i.e., single stranded nucleic acidsequences that capture complementary strand of target nucleic acid.) tocapture complementary strands of the amplified nucleic acid. Forexample, in some embodiments, the read lane can include a firstdetection surface that includes a hybridization probe specific toNeisseria gonorrhea (NG), a second detection surface that includes ahybridization probe specific to Chlamydia trachomatis (CT), and a thirddetection surface that includes a hybridization probe specific toTrichomonas vaginalis (TV).

In use, the post-amplification solution (from the outlet portion 5622 ofthe flow member 5610) is conveyed into the detection chamber 5812. Afterthe sample is in the detection chamber 5812, DNA strands in thepost-amplification solution can bind to certain detection surfaces tofacilitate production of the output signal. In some embodiments, tofacilitate such binding, the detection module 5800 and/or the detectionsurfaces therein are heated via the fifth heater assembly 5669 toincubate the amplicon within the read lane (e.g., in the presence of ahybridizing probe). The independently controllable and isolated fifthheater assembly 5669 allows for accurate control of the temperature ofthe detection chamber 5812, and also allows for the fifth heaterassembly 5669 to be activated at a different time than the other heaterassemblies (e.g., after the PCR is completed). After the ampliconhybridization has occurred, additional wash solutions and/or reagentscan be conveyed through the detection module 5800 to facilitate theproduction of an output signal. The fifth heater assembly 5669 can heatthe detection chamber 5812 (and the contents therein) at various stagesof the detection cycle to further facilitate and/or enhance theproduction of the output signal.

Referring to FIGS. 5 and 15, the heat sink 5690 is a thermallyconductive material (e.g., aluminum) that is coupled to the circuitboard assembly 5630 by a series of fasteners (not shown) that arecoupled within the mounting openings (or vias) 5639 defined by thecircuit board assembly 5630. The heat sink 5690 includes a series ofoffsets 5691 that are aligned with the openings 5639, and that maintainspacing between the heat sink 5690 and the flow member 5610. Thisarrangement allows for a consistent heat transfer path (or thermalcoupling) between the circuit board assembly 5630 and the heat sink 5690via the fasteners and the internal structure of the circuit boardassembly 5630.

The heat transfer between the circuit board assembly 5630 and the heatsink 5690 is further facilitated by a series of conductive layersbeneath and/or surrounding the second heater assembly 5660 and the thirdheater assembly 5670. The conductive layers provide a conductive path tofacilitate heat transfer away from the second heater assembly 5660 andthe third heater assembly 5670 to prevent overheating of the secondheating zone 5632 and the third heating zone 5633, respectively.Referring to FIG. 11, the first layer 5646 of the circuit board assembly5630 includes a first conductive region 5671 and a second conductiveregion 5672. The first conductive region 5671 (also referred to as acopper pour) surrounds, but is electrically isolated from the secondheater assembly 5660. The second conductive region 5672 (also referredto as a copper pour) surrounds, but is electrically isolated from thethird heater assembly 5670. The first conductive region 5671 and thesecond conductive region 5672 are thermally coupled to the mountingopenings 5639 (or vias), and thus provide a low resistance thermalconnection to the fasteners (not shown) that couple the printed circuitboard assembly 5630 to the heat sink 5690. In this manner, the firstconductive region 5671 and the second conductive region 5672 provide aconduction path to facilitate heat transfer away from the second heaterassembly 5660 and the third heater assembly 5670 to prevent overheatingof the second heating zone 5632 and the third heating zone 5633,respectively.

As shown in FIG. 12, the second layer 5647 of the circuit board assembly5630 includes a first conductive region 5673 and a second conductiveregion 5674. The first conductive region 5673 (also referred to as acopper pour) is beneath, but is electrically isolated from the secondheater assembly 5660. The second conductive region 5674 (also referredto as a copper pour) is beneath, but is electrically isolated from thethird heater assembly 5670. The first conductive region 5673 and thesecond conductive region 5674 are thermally coupled to the mountingopenings 5639 (or vias), and thus provide a low resistance thermalconnection to the fasteners (not shown) that couple the printed circuitboard assembly 5630 to the heat sink 5690. In this manner, the firstconductive region 5673 and the second conductive region 5674 provide aconduction path to facilitate heat transfer away from the second heaterassembly 5660 and the third heater assembly 5670 to prevent overheatingof the second heating zone 5632 and the third heating zone 5633,respectively. Further, in this embodiment, the portion of the secondlayer 5647 that is aligned with (or beneath) the first heater assembly5650 is devoid of a conductive material or copper pour. Because thefirst heater assembly 5650 is configured to operate at highertemperatures than either the second heater assembly 5660 or the thirdheater assembly 5670, an additional conduction path to facilitate heattransfer away from the first heating zone 5631 is not desired. In otherembodiments, however, any suitable layer of a printed circuit board caninclude conductive layers (or copper pour layers) beneath and/or alignedwith any of the heater assemblies and/or heating zones.

As shown in FIG. 13 the third layer 5648 of the circuit board assembly5630 includes a first conductive region 5675 and a second conductiveregion 5676. The first conductive region 5675 (also referred to as acopper pour) is beneath, but is electrically isolated from the secondheater assembly 5660. The second conductive region 5676 (also referredto as a copper pour) is beneath, but is electrically isolated from thethird heater assembly 5670. The first conductive region 5675 and thesecond conductive region 5676 are thermally coupled to the mountingopenings 5639 (or vias), and thus provide a low resistance thermalconnection to the fasteners (not shown) that couple the printed circuitboard assembly 5630 to the heat sink 5690. In this manner, the firstconductive region 5675 and the second conductive region 5676 provide aconduction path to facilitate heat transfer away from the second heaterassembly 5660 and the third heater assembly 5670 to prevent overheatingof the second heating zone 5632 and the third heating zone 5633,respectively. Further, in this embodiment, the portion of the thirdlayer 5648 that is aligned with (or beneath) the first heater assembly5650 is devoid of a conductive material or copper pour. Because thefirst heater assembly 5650 is configured to operate at highertemperatures than either the second heater assembly 5660 or the thirdheater assembly 5670, an additional conduction path to facilitate heattransfer away from the first heating zone 5631 is not desired. In otherembodiments, however, any suitable layer of a printed circuit board caninclude conductive layers (or copper pour layers) beneath and/or alignedwith any of the heater assemblies and/or heating zones.

As shown in FIG. 14, the fourth layer 5649 of the circuit board assembly5630 includes a first conductive region 5677 and a second conductiveregion 5678. The first conductive region 5677 (also referred to as acopper pour) is beneath, but is electrically isolated from the secondheater assembly 5660. The second conductive region 5678 (also referredto as a copper pour) is beneath, but is electrically isolated from thethird heater assembly 5670. The first conductive region 5677 and thesecond conductive region 5678 are thermally coupled to the mountingopenings 5639 (or vias), and thus provide a low resistance thermalconnection to the fasteners (not shown) that couple the printed circuitboard assembly 5630 to the heat sink 5690. In this manner, the firstconductive region 5677 and the second conductive region 5678 provide aconduction path to facilitate heat transfer away from the second heaterassembly 5660 and the third heater assembly 5670 to prevent overheatingof the second heating zone 5632 and the third heating zone 5633,respectively. Further, in this embodiment, the portion of the fourthlayer 5649 that is aligned with (or beneath) the first heater assembly5650 is devoid of a conductive material or copper pour. Because thefirst heater assembly 5650 is configured to operate at highertemperatures than either the second heater assembly 5660 or the thirdheater assembly 5670, an additional conduction path to facilitate heattransfer away from the first heating zone 5631 is not desired. In otherembodiments, however, any suitable layer of a printed circuit board caninclude conductive layers (or copper pour layers) beneath and/or alignedwith any of the heater assemblies and/or heating zones.

As shown in FIGS. 11-14, the conductive regions for each layer of theprinted circuit board assembly 5630 are discontinuous (segmented).Specifically, the first (or hot) heating zone 5631 is devoid of aconductive region, because the higher operating temperatures of thefirst heating zone 5631 do not necessitate a conductive region tofacilitate heat transfer away from the first heating assembly 5650.Similarly stated, the first heating zone 5631 is devoid of a conductiveregion to keep the thermal energy localized and to prevent it fromspilling over into the other heating zones. In other embodiments,however, a printed circuit board assembly can include one or moreconductive regions surrounding and/or beneath any of the heating zones.

Moreover, in some embodiments, one or more additional conductive regionscan be included in any layer of the printed circuit board assembly 5630to facilitate reduction of electromagnetic interference conveyed to anycomponents on the printed circuit board assembly 5630 (e.g. acontroller, a processor, or the like).

The printed circuit board assembly 5630 includes a series of temperaturesensors that provide feedback to enable control of the heater assembliesdescribed herein. Specifically, as shown in FIG. 14, the fourth layer5649 of the printed circuit board assembly 5630 includes a first seriesof temperature sensors 5680 disposed beneath each of the heatingelements of the first heater assembly 5650, a second series oftemperature sensors 5681 disposed beneath each of the heating elementsof the second heater assembly 5660, a third series of temperaturesensors 5682 disposed beneath each of the heating elements of the thirdheater assembly 5670, and a fourth temperature sensor 5683 disposedbeneath the fourth heater assembly 5668. The temperature sensors can beany suitable sensor, such as a thermistor, thermocouple or the like.Moreover, although the temperature sensors are shown as being fabricatedin the fourth layer 5649, in other embodiments, the temperature sensorscan be fabricated in any layer of the circuit board assembly 5630.

As shown in FIG. 11, the first layer 5646 of the printed circuit boardassembly 5630 includes a detection temperature sensor 5684 disposedbeneath the fifth heater assembly 5669. The detection temperature sensor5684 can be any suitable sensor, such as a thermistor, thermocouple orthe like. Moreover, although the detection temperature sensor 5684 isshown as being fabricated in the first layer 5646, in other embodiments,the temperature sensors can be fabricated in any layer of the circuitboard assembly 5630.

In some embodiments, the amplification module 5600 (and any of theamplification modules described herein) can include a power source (notshown) and a controller (not shown). The power source can be anysuitable power source to power the amplification module 5600. In someembodiments, power source can include an on-board AC converter thatreceives AC power and converts the power to a level suitable forsupplying current to the heater assemblies. In other embodiments, thepower source can be a DC power source (e.g., a battery) coupled to theprinted circuit board assembly 5630. For example, in some embodiments,the amplification module 5600 (and the diagnostic device within which itis disposed) is configured for a single use. In such embodiments, thepower source can have a capacity sufficient for only one test. In someembodiments, for example, the power source is a battery having a nominalvoltage of about 9 VDC and a capacity of less than about 1200 mAh. Inother embodiments, the power source can include multiple DC batteries,such as, for example, multiple 1.5 VDC cells (e.g., AAA or AA alkalinebatteries).

The controller can be coupled to the printed circuit board assembly5630, and can control the timing and amount of current supplied from thepower source to each of the heater assemblies included in the printedcircuit board assembly 5630. In some embodiments, the controller canalso include a temperature feedback module (not shown) that receivestemperature signal from the temperature sensors described above (e.g.,the temperature sensors 5680). The temperature feedback module includescircuitry, components, and/or code to produce a control signal that canfacilitate controlling current to the heater assemblies. In someembodiments, the controller 1500 can also include a flow module (notshown) that receives information associated with flow of the samplethrough the amplification module 5600 (and the diagnostic device withinwhich the amplification module 5600 is employed). The controller can becoupled to a computer (not shown) or other input/output device via theinput/output module (or interface).

The amplification module 5600 (and any of the amplification moduledescribed herein) can be used within any suitable diagnostic device,such as in any of the diagnostic devices shown and described inInternational Patent Publication No. WO2016/109691, entitled “Devicesand Methods for Molecular Diagnostic Testing,” which is incorporatedherein by reference in its entirety. One example of an integrated testdevice is shown in FIG. 18, which is a schematic block diagram of amolecular diagnostic system 6000 (also referred to as “system” or“diagnostic device”), according to an embodiment. The diagnostic device6000 is configured to manipulate a sample to produce an opticalindication associated with a target cell according to any of the methodsdescribed herein. In some embodiments, the diagnostic device 6000 can bea single-use, disposable device that can provide an optical outputwithout need for any additional instrument to manipulate or otherwisecondition the diagnostic device 6000. Said another way, the diagnosticdevice 6000 is an integrated cartridge/instrument, and the entire unitcan be used to perform a diagnostic assay and then be disposed. Thediagnostic device 6000 includes a sample transfer device 6100, a samplepreparation module 6200, an inactivation chamber 6300, a fluidic drivemodule 6400, a mixing chamber 6500, an amplification module 6600, areagent storage module 6700, a detection module 6800, apower/electronics module 6900, and a control module 6950. A briefdescription of the major subsystems of the diagnostic device 6000 isprovided below.

The sample transfer device 6100 is configured to transport a sample suchas, for example, a blood, urine, male urethral specimens, vaginalspecimens, cervical swab specimens, and/or nasal swab specimens samplegathered using a commercially available sample collection kit, to thesample preparation module 6200. The sample collection kit can be a urinecollection kit or swab collection kit. Non-limiting examples of suchsample collection kits include Copan Mswab or BD ProbeTec UrinePreservative Transport Kit, Cat #440928, neat urine. The sample transferdevice 6100 dispenses and/or otherwise transfers an amount of sample orsample/media to the sample preparation module 6200 through an input port(not shown). The input port can then be capped. In some embodiments, thesample transfer device 6100 can be locked and/or fixedly coupled to thesample preparation module 6200 as a part of the dispensing operation. Inthis manner, the interface between the sample transfer device 6100 andthe sample preparation module 6200 can be configured to prevent reuse ofthe diagnostic device 6000, transfer of additional samples, or the like.Although shown as including the sample transfer device 6100, in otherembodiments, the diagnostic device 6000 need not include a sampletransfer device.

In some embodiments, through a series of user actions or in anautomated/semi-automated matter, the sample preparation module 6200 isconfigured to process the sample. For example, the sample preparationmodule 6200 can be configured to concentrate and lyse cells in thesample, thereby allowing subsequent extraction of DNA. In someembodiments, the processed/lysed sample is pushed and/or otherwisetransferred from the sample preparation module 6200 to the inactivationchamber 6300, which is configured to inactivate, in the lysed sample,the proteins used during lysing. In some embodiments, the fluidic drivemodule 6400 is configured to aspirate the sample from the inactivationchamber 6300, and is further configured to convey the sample to theamplification module 6600. The fluidic drive module 6400 is alsoconfigured to convey the sample and/or reagents (e.g., from the reagentstorage module 6700) to perform any of the methods of diagnostic testingdescribed herein. Similarly stated, the fluidic drive module 6400 isconfigured to generate fluid pressure, fluid flow and/or otherwiseconvey the input sample through the modules of the device.

The mixing chamber 6500 mixes the output of inactivation chamber 6300with the reagents necessary to conduct a PCR reaction. In someembodiments, the mixing chamber 6500 can contain the PCR reagents in theform of one or more lyophilized reagent beads that contain the primersand enzymes necessary for PCR. In such embodiments, the mixing chamber6500 can be configured to hydrate and/or reconstitute the lyophilizedbeads in a given input volume, while ensuring even local concentrationsof reagents in the entirety of the volume. The mixing chamber 6500 caninclude any suitable mechanism for producing the desired solution, suchas, for example, a continuous flow mixing channel, an active mixingelement (e.g., a stir rod) and/or a vibratory mixing element. The mixedsample is then conveyed to the amplification module 6600 (e.g., by thefluidic drive module 6400).

The amplification module 6600 is configured to run polymerase chainreaction (PCR) on the sample to generate an amplified sample, in anymanner as described herein. For example, in some embodiments, theamplification module 6600 can be similar to the amplification module5600 (or any other amplification module described herein). After PCR,the amplified sample is further pushed, transferred or conveyed to adetection module 6800. In some embodiments, the detection module 6800 isconfigured to run and/or facilitate a colorimetric enzymatic reaction onthe amplified sample. In particular, a series of reagents from thereagent storage module 6700 can be conveyed by the fluidic drive module6400 to facilitate the optical output from the test. In someembodiments, the various modules/subsystems of the main diagnosticdevice 6000 are controlled and/or powered by the power/electronicsmodule 6900 and the control module 6950.

In some embodiments, the control module 6950 can include one or moremodules, and can automatically control the valves, pumps, power deliveryand/or any other components of the diagnostic device 6000 to facilitatethe molecular testing as described herein. The control module 6950 caninclude a memory, a processor, an input/output module (or interface),and any other suitable modules or software to perform the functionsdescribed herein.

Although the printed circuit board (or heater) assembly 5630 is shownand described as including a series of heater assemblies (i.e., thefirst heater assembly 5650, the second heater assembly 5660, and thethird heater assembly 5670) fabricated with and/or coupled to a first(or outer) layer of the printed circuit board assembly 5630, in otherembodiments, a printed circuit board (or heater) assembly can includeheater assemblies and/or heating elements within any suitable layer orportion of the circuit board. For example, in some embodiments, aprinted circuit board (or heater) assembly can include one or moreheating elements within an inner layer. Such an arrangement can, forexample, allow the thickness of lithographically produced heatingelements to be controlled and/or set to a desired value. This, in turn,can allow the resistance of the heating element to be set to a desiredvalue. Moreover, including the heating elements within an inner layercan reduce the amount of electromagnetic field (EMF) noise to which theother electronic components on the printed circuit board assembly (e.g.,processors and/or controllers) are exposed. Specifically, the placementof the heating elements within the printed circuit board assembly canlimit the amount of noise generated by the high return current (i.e.,“ground noise”) to which the processors and/or controllers are exposed.

Moreover, although the amplification assembly 5600 is shown anddescribed as including a single heat sink (i.e., the heat sink 5690), inother embodiments, any of the amplification assemblies described hereincan include any suitable heat sink and/or thermal managementarrangement.

For example, FIGS. 19-25 show various views of a molecular diagnostictest device 7000 (also referred to as a “test device” or “device”)within which any of the amplification modules described herein can beincluded. The test device 7000 can be similar to, and can contain any ofthe structure as, the molecular diagnostic test device 6000 or any othermolecular diagnostic test devices shown and described in InternationalPatent Publication No. WO2016/109691, entitled “Devices and Methods forMolecular Diagnostic Testing” (“the '691 PCT Application”) which isincorporated herein by reference in its entirety. In particular, thetest device 7000 is an integrated device (i.e., the modules arecontained within a single housing) that is suitable for use within apoint-of-care setting (e.g., doctor's office, pharmacy or the like),decentralized test facility, or at the user's home. In some embodiments,the device 7000 can have a size, shape and/or weight such that thedevice 7000 can be carried, held, used and/or manipulated in a user'shands (i.e., it can be a “handheld” device). In other embodiments, thetest device 7000 can be a self-contained, single-use device. Similarlystated, in some embodiments, the test device 7000 can be configured withlock-outs or other mechanisms to prevent re-use or attempts to re-usethe device.

Further, in some embodiments, the device 7000 can be a CLIA-waiveddevice and/or can operate in accordance with methods that are CLIAwaived. Similarly stated, in some embodiments, the device 7000 (and anyof the other devices shown and described herein) is configured to beoperated in a sufficiently simple manner, and can produce results withsufficient accuracy to pose a limited likelihood of misuse and/or topose a limited risk of harm if used improperly. In some embodiments, thedevice 7000 (and any of the other devices shown and described herein),can be operated by a user with minimal (or no) scientific training, inaccordance with methods that require little judgment of the user, and/orin which certain operational steps are easily and/or automaticallycontrolled. In some embodiments, the molecular diagnostic test device7000 can be configured for long term storage in a manner that poses alimited likelihood of misuse (spoilage of the reagent(s), expiration ofthe reagents(s), leakage of the reagent(s), or the like). In someembodiments, the molecular diagnostic test device 7000 is configured tobe stored for up to about 36 months, up to about 32 months, up to about26 months, up to about 24 months, up to about 20 months, up to about 18months, or any values there between.

The test device 7000 is configured to manipulate an input sample toproduce one or more output signals associated with a target cell,according to any of the methods described herein. FIGS. 10 and 11 showperspective views of the molecular diagnostic test device 7000. Thediagnostic test device 7000 includes a housing 7001 (including a topportion 7010 and a bottom portion 7030), within which a variety ofmodules are contained. Specifically, the device 7000 includes a samplepreparation module (not shown, but which is similar to the samplepreparation module 6200 described herein or in the '691 PCTApplication), an inactivation module (not shown, but which is similar tothe inactivation module 6300 described herein or in the '691 PCTApplication), a fluidic drive (or fluid transfer) module (not shown, butwhich is similar to the fluidic drive module 6400 described herein or inthe '691 PCT Application), a mixing chamber (not shown, but which issimilar to the mixing chamber 6500 described herein or in the '691 PCTApplication), an amplification module 7600, a detection module 7800, areagent storage module (not shown, but which is similar to the reagentstorage module 6700 described herein or in the '691 PCT Application),and a power and control module 7900.

The lower housing 7030 defines a volume 7032 within which the modulesand or components of the device 7000 are disposed. The top housing 7010covers and/or surrounds at least a portion of the lower housing 7030.FIG. 20 shows the device 7000 with the top housing 7010 removed so thatthe placement of the modules can be seen. In some embodiments, the tophousing 7010 can define a series of detection (or “status”) openingsthat allow the user to visually inspect the output signal(s) produced bythe device 7000. In such embodiments, when the top housing 7010 iscoupled to the lower housing 7030, the detection openings are alignedwith the corresponding detection surfaces of the detection module 7800such that the signal produced by and/or on each detection surface isvisible through the corresponding detection opening.

Although not described in detail herein, the sample preparation moduleincludes any or all of a sample input portion (or module), a washportion (or module), an elution portion (or module), a filter portion(or module), and various fluidic conduits (e.g., tubes, lines, valves,etc.) connecting the various components. The input sample can beconveyed into the test device 7000 by moving the cap 7152 and depositingthe sample into the input opening 7162. The sample preparation module isat least partially actuated by depressing the first actuator (or button)7050. In some embodiments, the first actuator 7050 includes tabs, locks,or the like that prevent the user from reusing the first actuator 7050and/or the device 7000 after an initial use has been attempted and/orcompleted. Further aspects of the sample preparation module (e.g., theelution portion or the filter portion) can be actuated by depressing thesecond actuator (or button) 7070. In some embodiments, the secondactuator 7070 includes tabs, locks, or the like that prevent the userfrom reusing the second actuator 7070 and/or the device 7000 after aninitial use has been attempted and/or completed. The reagent storagemodule and/or other aspects of the test device 7000 (including the powerand control module 7900) can be actuated by depressing the thirdactuator (or button) 7080. In some embodiments, the third actuator 7080includes tabs, locks, or the like that prevent the user from reusing thethird actuator 7080 and/or the device 7000 after an initial use has beenattempted and/or completed. Moreover, in some embodiments, the firstactuator 7050, the second actuator 7070, and the third actuator caninclude tabs, locks, or other structure to control the order ofoperation of the actuators.

The amplification module 7600 is configured to perform a thermalreaction (e.g., an amplification reaction) on an input of target nucleicacid mixed with required reagents. Referring to FIG. 22, theamplification module 7600 includes a flow member 7610, a circuit board(or heater) assembly 7630, a first (or lower) heat sink 7690, and asecond (or upper) heat sink 7692. The flow member 7610 is coupledbetween the circuit board assembly 7630 and the first heat sink 7690.The flow member 7610 has the same structure and function as the flowmember 6610 described above, and is therefore not described in detailbelow. In particular, the flow member defines a flow path 7620 throughwhich a sample can flow from an inlet port to an outlet port. The flowmember 7620 defines a flow axis A_(F) that indicates the overalldirection of the flow through the flow member 7610. As shown, theamplification flow path has a curved, switchback or serpentine pattern.More specifically, the flow member (or chip) 7610 has two serpentinepatterns—an amplification pattern and a hot-start pattern 7623. Theamplification pattern allows for amplification (i.e., PCR in thisinstance) to occur while the hot-start pattern 7623 accommodates thehot-start conditions of the PCR enzyme.

As shown, the serpentine pattern establishes 40 different zones of“cold-to-hot-to-cold;” or 40 amplification cycles. In other embodiments,however, the flow member 7610 (or any of the other flow membersdescribed herein) can define any suitable number of switchbacks oramplification cycles to ensure the desired test sensitivity. In someembodiments, the flow member can define at least 30 cycles, at least 34cycles, at least 36 cycles, at least 38 cycles, or at least 40 cycles.The dimensions of the flow channel 7620 in the flow member 7610 impactthe temperature conditions of the PCR and dictate the overall dimensionsof the chip, and thus affect the overall power consumption of theamplification module 7600. For example, a deeper, narrower channel willdevelop a larger gradient in temperature from the side closest to thelid 7619 to the bottom (resulting in lower PCR efficiency). Thisarrangement, however, requires less overall space since the channelswill take up less overall surface area facing the heater assembly 7630(and thus require less energy to heat). The opposite holds true for awide and shallow channel. In some embodiments, the depth of the flowchannel 7620 is about 0.15 mm and the width of the flow channel 7620 isbetween about 1.1 mm and about 1.3 mm. More particularly, in someembodiments, the flow channel 7620 has a width of about 1.1 mm in the“narrow” sections (that are within the second temperature zone 7612 andthe third temperature zone 7613) and about 1.3 mm in the “wide” section(that falls within the first temperature zone 7611). In someembodiments, the overall path length is about 960 mm (including both theamplification portion and the hot start portion 7623). In suchembodiments, the total path length of the amplification portion is about900 mm. This produces a total volume of the flow channel 7620 of about160 μl (including the hot start portion 7623) and about 150 μl (withoutthe hot start portion 7623). In some embodiments, the separation betweeneach parallel path is between about 0.4 mm and about 0.6 mm.

The flow member 7610 can be constructed from any suitable material, andcan have any suitable thickness. For example, in some embodiments, theflow member 7610 (and any of the flow members described herein) can bemolded from COC (Cyclic Olefin Copolymer) plastic, which has inherentbarrier properties and low chemical interactivity. In other embodiments,the flow member 7610 (and any of the flow members described herein) canbe constructed from a graphite-based material (for improved thermalproperties). The overall thickness of the flow member 7610 can be lessthan about 0.5 mm, less than about 0.4 mm, less than about 0.3 mm orless than about 0.2 mm.

The flow member 7610 can be coupled to the circuit board assembly 7630in any suitable manner. For example, in some embodiments, the flowmember 7610 can be coupled to the heater assembly 7630 at least in partby the mechanical fasteners 7691 used to couple the first heat sink 7690and the second heat sink 7692 to the circuit board assembly 7630. Insome such embodiments, the fasteners 7691 can also function as heatsinks (or conduits) to allow accurate control of the temperatures of theflow member 7610 and to avoid overheating. In other embodiments, theflow member 7610 can be coupled to the heater assembly 7630 by anadhesive (e.g., a pressure-sensitive adhesive). Similarly stated, insome embodiments, the flow member 7610 can be chemically bonded to theheater assembly 7630. Thus, the flow member 7610 can be fixedly andirreversibly coupled to the heater assembly 7630. Said another way, insome embodiments, the flow member 7610 is not designed to be removedand/or decoupled from the heater assembly 7630 during normal use. Thisarrangement facilitates the test device 7000 being a single-use,disposable device.

The circuit board (or heater) assembly 7630 is a multi-layer circuitboard having a first side 7637 and a second side 7638. FIG. 24 shows anexploded view of various layers of the circuit board assembly 7630,including certain copper traces fabricated thereon. Although FIG. 24shows a substrate and three layers, in some embodiments, a thin filmsubstrate (not shown) separates adjacent layers. In other embodiments, acircuit board assembly can include any suitable number of layers. Forexample, the circuit board assembly 7630 (and any other circuit boardassemblies described herein) can include one or more layers havingconductive regions similar to the conductive regions 5671, 5672 shownand described above with reference to the circuit board assembly 5630.

Specifically, at least the second (or outer bottom) layer 7647 includesa first conductive region 7675, a second conductive region 7673, and athird conductive region 7674. The first conductive region 7675 (alsoreferred to as a copper pour) is beneath, but is electrically isolatedfrom the first heater assembly 7650. The inclusion of the firstconductive region 7675 is different than the design shown above for theamplification module 5600, in which the portion of the second layer 5647aligned with (or beneath) the first heater assembly 5650 is devoid of aconductive material or copper pour. Here, the first conductive region7675 is isolated from the other conductive regions, and providesaccurate control of the temperatures in the first region. The secondconductive region 7673 (also referred to as a copper pour) is beneath,but is electrically isolated from the second heater assembly 7660. Thethird conductive region 7674 (also referred to as a copper pour) isbeneath, but is electrically isolated from the third heater assembly7670. The second conductive region 7673 and the third conductive region7674 are thermally coupled to the mounting openings, and thus provide alow resistance thermal connection to the fasteners (not shown) thatcouple the printed circuit board assembly 7630 to the first heat sink7690 and/or the second heat sink 7692. In this manner, the conductiveregions provide a conduction path to facilitate heat transfer away fromthe first heater assembly 7650, the second heater assembly 7660 and thethird heater assembly 7670.

Moreover, in addition to including the heater assemblies as describedherein, the circuit board assembly 7630 is also coupled to and/orsupports the processor 7950, and other electronic components of thepower and control module 7900. Thus, the circuit board (or heater)assembly 7630 performs and/or facilitates the performance of manydifferent electronic functions, including controlling the amplificationof the sample, controlling sample movement, and other thermally-basedfunctions described herein.

As shown, the circuit board assembly 7630 includes a substrate 7640having a first side 7657 and a second side 7658, a first (or heater)layer 7646, a second (or outer bottom) layer 7647, and a third (or outertop) layer 7648. The substrate 7640 provides structural support, and isconstructed from an electrically isolative material upon which the fourlayers are fabricated using lithographic procedures. The substrate 7640(and any of the substrates described herein) can be constructed from anysuitable material, such as, for example, a composite material includingwoven glass and epoxy. In some embodiments, the substrate 7640 (and anyof the substrates described herein) can be constructed from a materialhaving a glass transition temperature (Tg) of greater than about 170 C.In other embodiments, the substrate 7640 (and any of the substratesdescribed herein) can be constructed from a material having a glasstransition temperature (Tg) of greater than about 180 C (e.g., material370HR produced by the Isola Group). In this manner, the substrate 7640can maintain the desired rigidity and dimensional integrity to providerepeatable thermal performance for each channel of the flow path 7620.

Referring again to FIG. 23, the circuit board assembly 7630 defines aseries of apertures (also referred to as openings, cut-outs, or vias)that separate the circuit board assembly 7630 into several differentportions (or heating zones). Specifically, the circuit board assembly7630 defines a first set of apertures 7641 that separates a firstportion (or heating zone) 7631 of the assembly 7630 from a secondportion (or heating zone) 7632 of the assembly 7630. The first set ofapertures 7641 includes three openings that are elongated along the flowaxis A_(F), and that are separated by two connection lugs 7651. Thus,the first set of apertures 7641 produces a longitudinally orientedthermal barrier between the first heating zone 7631 and the secondheating zone 7632. Similarly, the circuit board assembly 7630 defines asecond set of apertures 7642 that separates the first portion (orheating zone) 7631 of the assembly 7630 from a third portion 7633 (orheating zone) of the assembly 7630. The second set of apertures 7642includes three openings that are elongated along the flow axis A_(F),and that are separated by two connection lugs 7652. Thus, the second setof apertures 7642 produces a longitudinally oriented thermal barrierbetween the first heating zone 7631 and the third heating zone 7633.

The first heating zone 7631 is disposed between the second heating zone7632 and the third heating zone 7633. Moreover, like the arrangement ofthe circuit board assembly 5630 described above, when the circuit boardassembly 7630 is coupled to the flow member 7610, the first heating zone7631 is aligned with a first (or “hot”) temperature zone of the flowmember 7610, the second heating zone 7632 is aligned with a second (or“cold”) temperature zone of the flow member 7610, and the third heatingzone 7633 is aligned with a third (or “cold”) temperature zone of theflow member 7610. Referring to FIG. 25, this arrangement allows thefirst heater assembly 7650 located within first heating zone 7631 toheat the first temperature zone (or central portion) of the flow member7610. This arrangement further allows the second heater assembly 7660located within second heating zone 7632 to heat the second temperaturezone (or side portion) of the flow member 7610. This arrangement furtherallows the third heater assembly 7670 located within third heating zone7633 to heat the third temperature zone (or opposite side portion) ofthe flow member 7610. In this manner, the heater assembly 7630 and theflow member 7610 can establish multiple temperature zones through whicha sample can flow, and can define a desired number of amplificationcycles to ensure the desired test sensitivity (e.g., at least 30 cycles,at least 34 cycles, at least 36 cycles, at least 38 cycles, or at least40 cycles).

As shown in FIG. 25, the first heater assembly 7650 includes a firstheating element 7661, a second heating element 7662, and a third heatingelement 7663, each of which is electrically isolated from the other twoheating elements in the first heater assembly 7650. Said another way,each of the first heating element 7661, the second heating element 7662,and the third heating element 7663 is separate from (or electricallyisolated from) the others. In this manner, an electrical current can beconveyed to each of the first heating element 7661, the second heatingelement 7662, and the third heating element 7663 independently from anelectrical current being conveyed to the other heating elements of thefirst heater assembly 7650. This arrangement allows for independentcontrol of the first heating element 7661, the second heating element7662, and the third heating element 7663. The second heater assembly7660 includes a first heating element 7664 and a second heating element7665, each being electrically isolated from the other. Said another way,the first heating element 7664 is separate from the second heatingelement 7665, and vice-versa. In this manner, an electrical current canbe conveyed to the first heating element 7664 independently from anelectrical current being conveyed to the second heating element 7665,and vice-versa. This arrangement allows for independent control of thefirst heating element 7664 and the second heating element 7665. Thethird heater assembly 7670 includes a first heating element 7666 and asecond heating element 7667, each being electrically isolated from theother. Said another way, the first heating element 7666 is separate fromthe second heating element 7667, and vice-versa. In this manner, anelectrical current can be conveyed to the first heating element 7666independently from an electrical current being conveyed to the secondheating element 7667, and vice-versa. This arrangement allows forindependent control of the first heating element 7666 and the secondheating element 7667.

Each of the heating elements described above are conductive traces thatare fabricated on the heater layer 7646 by lithographic techniques. Thefirst layer 7646 is between the outer bottom-most layer 7647. Byarranging the heater layer 7646 within the overall circuit board, theeffect of any EMF noise generated by the current supply or return to theheaters on the processor 7950 can be minimized. In other embodiments,the first heater assembly 7650 can be fabricated in any layer of thecircuit board assembly 7630.

In use, the first heater assembly 7650 produces a thermal output tomaintain the first temperature zone of the flow member 7610 at a firsttemperature. The first temperature can be, for example, between about100 C and 115 C (to heat the sample therein to about 90 C; e.g., the“hot” temperature for a PCR thermal cycle). Additionally, the segmented,independently controllable design allows the first heating element 7661to produce a first thermal output, the second heating element 7662 toproduce a second thermal output, and the third heating element 7663 toproduce a third thermal output, each of which can be different from theothers. By producing different thermal outputs, the hot portion of theflow channel 7620 can be more accurately maintained at the desiredtemperature. The second heater assembly 7660 produces a thermal outputto maintain the second temperature zone of the flow member 7610 at asecond temperature. The second temperature can be different from thefirst temperature, and can be, for example, about 60 C to about 75 C (toheat the sample therein to about 60 C; e.g., the “cold” temperature fora PCR thermal cycle). Additionally, the segmented, independentlycontrollable design allows the first heating element 7664 to produce afirst thermal output and the second heating element 7665 to produce asecond thermal output different from the first thermal output. Byproducing different thermal outputs, the cold portion of the flowchannel 7620 can be more accurately maintained at the desiredtemperature. The third heater assembly 7670 produces a thermal output tomaintain the third temperature zone 7613 of the flow member 7610 at athird temperature. The third temperature can be different from the firsttemperature and/or the second temperature. In some embodiments, thethird temperature can be the same as the second temperature, and can be,for example, about 60 C to about 75 C (to heat the sample therein toabout 60 C; e.g., the “cold” temperature for a PCR thermal cycle).Additionally, the segmented, independently controllable design allowsthe first heating element 7666 to produce a first thermal output and thesecond heating element 7667 to produce a second thermal output differentfrom the first thermal output. By producing different thermal outputs,the cold portion of the flow channel 7620 can be more accuratelymaintained at the desired temperature.

In some embodiments, the sample flowing within the flow path 3620 israpidly heated to about 90 C. To promote a rapid cooling down to about60 C, in some embodiments, heat must flow out of the sample (and thusthe flow member 7610). Thus, although the second temperature isdescribed as being hotter than the desired sample temperature, in otherembodiments, the output produced by the second heater assembly 7660and/or the third heater assembly 7670 (or any of the heating elementsdescribed herein) can be such that heat flows out of the flow path 7620and/or the flow member 7610. In such embodiments, a current can still besupplied to the second heater assembly 7660 and/or the third heaterassembly 7670 to control the magnitude of the heat flow. In someembodiments, the second temperature and/or the third temperature can be,for example, between about 40 C and about 45 C (to allow heat transferaway from the sample at a controlled rate to facilitate maintaining thesample at about 60 C; e.g., the “cold” temperature for a PCR thermalcycle).

As described above, the first set of apertures 7641 produces alongitudinally oriented thermal barrier between the first heating zone7631 and the second heating zone 7632, and the second set of apertures7642 produces a longitudinally oriented thermal barrier between thefirst heating zone 7631 and the third heating zone 7633. Thus, the firstset of apertures 7641 and the second set of apertures 7642 collectivelythermally isolate the first heating zone 7631 of the circuit boardassembly 7630. By minimizing the heat transfer between the first heatingzone 7631, the second heating zone 7632, and the third heating zone7633, accuracy and control of the temperature to which each heating zoneis heated can be improved.

Referring to FIG. 23, the connection lugs (or portions) 7651 thatseparate the first set of apertures 7641 into three openings are offsetfrom the connection lugs (or portions) 7652 that separate the second setof apertures 7642 into three openings. Similarly stated, a connectionlug 7651 is located at a different longitudinal position than acorresponding connection lug 7652. Said another way, the connection lugs7651 are positioned at a different location along the flow axis A_(F)than are the connection lugs 7652. This arrangement allows each of theconnection lugs 7651 and the connection lugs 7652 to be positioned below(or aligned with) a different channel of the flow path 7620 when thecircuit board assembly 7630 is coupled to the flow member 7610. As anexample, this arrangement allows a connection lug 7651 to be alignedwith, for example, the tenth channel within the flow path 7620 while thecorresponding connection lug 7652 is aligned with, for example, theeighteenth channel within the flow path 7620. Because the thermalperformance of the first heating zone 7631 in the areas adjacent theconnection lugs 7651 and 7652 is different than the thermal performanceat other spatial locations, the offset arrangement of the connectionlugs minimizes any differences in the temperature of the flow channel.This, in turn, increases the overall accuracy of the device.

In addition to including three heating zones for the PCR reaction, thecircuit board assembly 7630 also defines a third aperture 7643 thatseparates a fourth portion (or heating zone) 7634 from the other heatingzones. The third aperture 7643 is elongated substantially perpendicularto the flow axis A_(F), and thus produces a laterally-oriented thermalbarrier between the fourth heating zone 7634 and the amplificationheating zones (i.e., the first heating zone 7631, the second heatingzone 7632, and the third heating zone 7633). Although shown as being asingle opening, in other embodiments the fourth heating zone 7634 can beseparated by a series of apertures and connection lugs.

As shown, the fourth heating zone 7634 is disposed at an end portion ofthe circuit board assembly 7630. When the circuit board assembly 7630 iscoupled to the flow member 7610, the fourth portion 7634 is aligned withthe hot-start pattern 7623 of the flow member 7610. This arrangementallows the fourth heater assembly 7668 located within fourth heatingzone 7634 to heat the hot-start pattern 7623 of the flow member 7610.The hot-start portion 7623 reduces non-specific amplification and allowsthe use of certain PCR reagents that remain inactive until heated. Insome embodiments, the first heater assembly 7650 can be controlled tomaintain the first temperature zone 7611 at a temperature of betweenabout 45 degrees Celsius and about 95 degrees Celsius (and/or at asurface temperatures such that the fluid flowing therethrough reaches atemperature between about 45 degrees Celsius and about 95 degreesCelsius).

The fourth heating zone 7634 of the printed circuit board assembly 7630includes a fourth heater assembly 7668. The fourth heater assembly 7668includes a single heating element that is electrically isolated from theheating elements included in the other heating assemblies (e.g., thefirst heating assembly 7650). In this manner, an electrical current canbe conveyed to the fourth heater assembly 7668 independently from anelectrical current being conveyed to the other heating elements of thefirst heater assembly 7650, the second heater assembly 7660 and/or thethird heater assembly 7670. This arrangement allows for independentcontrol of the hot-start portion of the amplification module 7600.

Unlike the detection module 5800, the detection module 7800 is notcoupled directly to the printed circuit board assembly 7630. Instead thedetection module 7800 is coupled to (either directly or via interveningstructure), the second heat sink 7692. In some embodiments, for example,the detection module 7800 can be coupled to a detection heater (notshown) that is mounted to the second heat sink 7692, and that heatsportions of the detection module 7800. In other embodiments, thedetection module 7800 can be coupled directly to the second heat sink7692, which provides the desired heat (via the heater assemblies withinthe circuit board assembly 7630) to heat portions of the detectionmodule 7800. In yet other embodiments, the detection module 7800 can becoupled to the second heat sink 7692 in a manner such that an air gapbetween the detection module 7800 and the second heat sink 7692 isproduced.

The detection module 7800 is configured to receive output from theamplification module 7600 (i.e., from the flow member 7610) and reagentsfrom a reagent module (not shown) to produce an output to indicatepresence or absence of target organism in the initial input sample. Thedetection module 7800 can also produce an output to indicate the generalcorrect operation of the test (positive control and negative control).For example, in some embodiments, the detection module 7800 can produceone or more colorimetric outputs. The detection module 6800 can be anyof the detection modules shown and described in International PatentPublication No. WO2016/109691, entitled “Devices and Methods forMolecular Diagnostic Testing,” which is incorporated herein by referencein its entirety.

The detection module 7800 includes a detection flow cell 7810 thatdefines a detection chamber/channel 7812 having a series of inlet andoutlet portions, through which the sample and the reagents are conveyedto produce the detected output. The detection chamber 7812 includes a“read lane” 7820 (also referred to as a detection portion) that includesone or more detection surfaces or zones. In some embodiments, thedetection surfaces can be chemically modified to contain hybridizationprobes (i.e., single stranded nucleic acid sequences that capturecomplementary strand of target nucleic acid.) to capture complementarystrands of the amplified nucleic acid. For example, in some embodiments,the read lane can include a first detection surface that includes ahybridization probe specific to Neisseria gonorrhea (NG), a seconddetection surface that includes a hybridization probe specific toChlamydia trachomatis (CT), and a third detection surface that includesa hybridization probe specific to Trichomonas vaginalis (TV).

In use, the post-amplification solution (from the outlet portion 7622 ofthe flow member 7610) is conveyed into the detection chamber 7812. Afterthe sample is in the detection chamber 7812, DNA strands in thepost-amplification solution can bind to certain detection surfaceswithin the read lane 7820 to facilitate production of the output signal.In some embodiments, to facilitate such binding, the detection module7800 and/or the detection surfaces therein are heated to incubate theamplicon within the read lane (e.g., in the presence of a hybridizingprobe). As described above, the detection module 7800 can be heatedeither by a separate heater or by the second heat sink 7692.

FIG. 26 is a flow chart of a method 10 of heating a sample (e.g., toamplify a nucleic acid therein) according to an embodiment. The method10 can be performed using any suitable device, such as the amplificationmodule 5600 or the amplification module 7600 described herein. Themethod includes conveying a sample into a diagnostic device, at 12. Thediagnostic device includes a flow member coupled to a first heaterassembly (e.g., the heater 1650) and a second heater assembly (e.g., theheater 1660). The flow member can be any suitable flow member (e.g., theflow member 1610) defining a flow path having a set of flow channels. Insome embodiments, the first heater assembly includes a first heatingelement (e.g., the heating element 5661) and a second heating element(e.g., the heating element 5662). The second heater assembly includes athird heating element (e.g., the heating element 5664) and a fourthheating element (e.g., the heating element 5665).

The method 10 includes actuating the diagnostic device, at 13. Uponactuation, a current is supplied, at a first time, to the first heatingelement and the third heating element, at 13A. The current is suppliedsuch that the first heating element maintains at least a first portionof a first channel from the set of channels at a first temperature, andthe third heating element maintains at least a second portion of thefirst channel from the set of channels at a second temperature. Inresponse to the actuation, a flow of sample within the flow path isproduced at a second time, at 13B. The second time occurs after thefirst time. In this manner, the method 10 provides for the first heatingelement and the third heating element to be activated before the flow ofsample is introduced. In response to the actuation, a current issupplied, at a third time, to the second heating element and the fourthheating element, at 13C. The current is supplied such that the secondheating element maintains at least a first portion of a second channelfrom the set of channels at the first temperature, and the fourthheating element maintains at least a second portion of the secondchannel from the set of channels at the second temperature. The thirdtime is different from the first time. In this manner, the second andfourth heating elements, which supply heat to subsequent flow channels(e.g., the last flow channel) are activated at a later time, thusconserving power and/or minimizing peak power usage.

FIG. 27 is a flow chart of a method 20 of heating a sample (e.g., toamplify a nucleic acid therein), according to an embodiment. The method20 can be performed using any suitable device, such as the amplificationmodule 5600 or the amplification module 7600 described herein. Themethod includes conveying a sample into a diagnostic device, at 22. Thediagnostic device, which can be the device 7000, includes a flow membercoupled to a heater assembly. The flow member can be any of the flowmembers described herein, and defines a flow path. The heater assembly(or printed circuit board assembly) includes a substrate, a firstheating element, and a second heating element. The heater assembly iscoupled to the flow member such that the first heating element isbetween a first portion of the substrate and a first portion of the flowpath, and the second heating element is between a second portion of thesubstrate and a second portion of the flow path. A third portion of thesubstrate separates the first portion of the substrate and the secondportion of the substrate. The third portion of the substrate ischaracterized by a thermal conductivity that is less than a thermalconductivity of the first portion of the substrate.

The method 20 includes actuating the diagnostic device, at 23. Uponactuation, a first current is supplied to the first heating element suchthat the first heating element maintains the first portion of the flowpath at a first temperature, at 23A. A second current is supplied to thesecond heating element such that the second heating element maintainsthe second portion of the flow path at a second temperature, at 23B. Thesecond temperature different from the first temperature. A flow ofsample within the flow path is produced, at 23C.

FIG. 28 is a flow chart of a method 30 of heating a sample (e.g., toamplify a nucleic acid therein), according to an embodiment. The method30 can be performed using any suitable device, such as the amplificationmodule 5600 or the amplification module 7600 described herein. Themethod includes conveying a sample into a diagnostic device, at 32. Thediagnostic device, which can be the device 7000, includes a flow membercoupled to a first heater assembly and a second heater assembly. Theflow member defines a flow path having a series of flow channels. Thefirst heater assembly includes a first heating element. The secondheater assembly includes a second heating element and a third heatingelement. The first heater assembly is coupled to the flow member suchthat the first heating element is aligned with a first portion of theflow path. The second heater assembly is coupled to the flow member suchthat and the second heating element and the third heating element areeach aligned with a second portion of the flow path.

The method 30 includes actuating the diagnostic device, at 33. Uponactuation, a first current is supplied to the first heating element suchthat the first heating element maintains the first portion of the flowpath at a first temperature, at 33A. A flow of sample within the flowpath is produced, at 33B. A second current is supplied to the secondheating element, at 33C. A third current is supplied to the thirdheating element, at 33D. The third current is supplied independentlyfrom the second current. The second current and the third current aresupplied such that the second heating element and the third heatingelement collectively maintain the second portion of the flow path at asecond temperature.

While various embodiments have been described above, it should beunderstood that they have been presented by way of example only, and notlimitation. Where methods and/or schematics described above indicatecertain events and/or flow patterns occurring in certain order, theordering of certain events and/or flow patterns may be modified. Whilethe embodiments have been particularly shown and described, it will beunderstood that various changes in form and details may be made.

For example, any of the amplification modules, heater assemblies, anddetection modules shown and described herein can be used in any suitablediagnostic device. Such devices can include, for example, a single-usedevice that can be used in a point-of-care setting and/or in a user'shome. Similarly stated, in some embodiments, the device (and any of theother devices shown and described herein) can be configured for use in adecentralized test facility. Further, in some embodiments, any of theamplification modules, heater assemblies, and detection modules shownand described herein can be included within a CLIA-waived device and/orcan facilitate the operation of a device in accordance with methods thatare CLIA waived. Similarly stated, in some embodiments, theamplification modules, heater assemblies, and detection modules shownand described herein can facilitate operation of a device in asufficiently simple manner that can produce results with sufficientaccuracy to pose a limited likelihood of misuse and/or to pose a limitedrisk of harm if used improperly. In some embodiments, the amplificationmodules, heater assemblies, and detection modules shown and describedherein can be used in any of the diagnostic devices shown and describedin International Patent Publication No. WO2016/109691, entitled “Devicesand Methods for Molecular Diagnostic Testing,” which is incorporatedherein by reference in its entirety,” which is incorporated herein byreference in its entirety.

The devices and methods described herein, however, are not limited toperforming a molecular diagnostic test on human samples. In someembodiments, any of the devices and methods described herein can be usedwith veterinary samples, food samples, and/or environmental samples.

Although the substrates (e.g. the substrate 1640, 2640, 3640, 4640,5640, and others) are shown and described herein as being rigid andhaving a glass transition temperature (Tg) of at least about 170 degreesCelsius, in other embodiments, an amplification module can include asubstrate (e.g. the substrate 1640, 2640, 3640, 4640, 5640, and others)having an suitable glass transition temperature (Tg), such as a Tg of aslow as 120 degrees Celsius or 100 degrees Celsius. In other embodiments,an amplification module can include a substrate (e.g. the substrate1640, 2640, 3640, 4640, 5640, and others) having a flexible substrate.For example, in some embodiments, an amplification module can include asubstrate constructed from any of Pyralux®, Nikaflex®, or Kapton®.

Although the first heater assembly 5650 is shown and described asincluding three independently controllable heating elements, in otherembodiments, any of the heater assemblies shown and described herein caninclude any suitable number of independently controllable heatingelements.

Although the heater assembly 5630 is shown and described as include afirst connection lug (or portion) 5651 that is longitudinally offsetfrom a second connection lug (or portion) 5652 by a distance equal tothat of one or two flow channels, in other embodiments, a heaterassembly can include a first connection lug that is longitudinallyoffset from a second connection lug by any suitable distance, such asfor example, by a distance equal to about 4 flow channels, between 4 and6 flow channels, between 6 and 8 flow channels, between 8 and 10 flowchannels, and between 10 and 12 flow channels.

In some embodiments, any of the amplification modules described can beconfigured to conduct a “rapid” PCR (e.g., completing at least 30 cyclesin less than about 10 minutes), and rapid production of an output signal(e.g., via a detection module). Similarly stated, the amplificationmodules described herein can be configured to process volumes, to havedimensional sizes and/or be constructed from materials that facilitatesa rapid PCR or amplification in less than about 10 minutes, less thanabout 9 minutes, less than about 8 minutes, less than about 7 minutes,less than about 6 minutes, or any range therebetween, as describedherein.

Some embodiments described herein relate to a computer storage productwith a non-transitory computer-readable medium (also can be referred toas a non-transitory processor-readable medium) having instructions orcomputer code thereon for performing various computer-implementedoperations. The computer-readable medium (or processor-readable medium)is non-transitory in the sense that it does not include transitorypropagating signals per se (e.g., a propagating electromagnetic wavecarrying information on a transmission medium such as space or a cable).The media and computer code (also can be referred to as code) may bethose designed and constructed for the specific purpose or purposes.Examples of non-transitory computer-readable media include, but are notlimited to: magnetic storage media such as hard disks, floppy disks, andmagnetic tape; optical storage media such as Compact Disc/Digital VideoDiscs (CD/DVDs), Compact Disc-Read Only Memories (CD-ROMs), andholographic devices; magneto-optical storage media such as opticaldisks; carrier wave signal processing modules; and hardware devices thatare specially configured to store and execute program code, such asApplication-Specific Integrated Circuits (ASICs), Programmable LogicDevices (PLDs), Read-Only Memory (ROM) and Random-Access Memory (RAM)devices.

Examples of computer code include, but are not limited to, micro-code ormicroinstructions, machine instructions, such as produced by a compiler,code used to produce a web service, and files containing higher-levelinstructions that are executed by a computer using an interpreter. Forexample, embodiments may be implemented using imperative programminglanguages (e.g., C, Fortran, etc.), functional programming languages(Haskell, Erlang, etc.), logical programming languages (e.g., Prolog),object-oriented programming languages (e.g., Java, C++, etc.) or othersuitable programming languages and/or development tools. Additionalexamples of computer code include, but are not limited to, controlsignals, encrypted code, and compressed code.

The processor 7950 (and any of the processors and/or controllersdescribed herein) can be any processor configured to, for example, writedata into and read data from the memory of the controller, and executethe instructions and/or methods stored within the memory. Furthermore,the processor can be configured to control operation of the othermodules within the controller (e.g., the temperature feedback module andthe flow module). Specifically, the processor can receive a signalincluding temperature data, current measurements or the like anddetermine an amount of power and/or current to be supplied to eachheater assembly, the desired timing and sequence of the piston pulsesand the like. For example, in some embodiments, the controller can be an8-bit PIC microcontroller, which will control the power delivered tovarious heating assemblies and components within the amplificationmodule 5600. This microcontroller can also contain code for and/or beconfigured to minimize the instantaneous power requirements on the powersource. The highest power consumption can occur, for example, whenamplification heaters (e.g. the first heating assembly 5650, the secondheating assembly 5660, and the third heating assembly 5670) are beingraised to temperature. By scheduling these warmup times during periodsof low power consumption of other portions of the device within whichthe amplification module 5600 is employed, the power requirements on thepower source can be reduced at the expense of increased energyconsumption. When multiple loads require power simultaneously, thecontroller contains code for and/or is configured to ensure that eachload receives the necessary average power while minimizing the time inwhich multiple loads are powered simultaneously. This is achieved byinterleaving the PWM signals to each load such that the periods in whichboth signals are in an on state is kept to a minimum.

In other embodiments, the processor (and any of the processors describedherein) can be, for example, an application-specific integrated circuit(ASIC) or a combination of ASICs, which are designed to perform one ormore specific functions. In yet other embodiments, the microprocessorcan be an analog or digital circuit, or a combination of multiplecircuits.

The memory device of the controller (and any of the memory devicesdescribed herein) can be any suitable device such as, for example, aread only memory (ROM) component, a random access memory (RAM)component, electronically programmable read only memory (EPROM),erasable electronically programmable read only memory (EEPROM),registers, cache memory, and/or flash memory. Any of the modules (thepressure feedback module and the position feedback module) can beimplemented by the processor and/or stored within the memory.

Although various embodiments have been described as having particularfeatures and/or combinations of components, other embodiments arepossible having a combination of any features and/or components from anyof embodiments as discussed above.

For example, although the substrate 4640 is not shown as defining anaperture, in other embodiments, the substrate 4640 (or any of the othersubstrates shown or described herein) can define an aperture similar tothe aperture 1641 shown and described with reference to theamplification module 1600.

As another example, although the circuit board assembly 5630 is shownand described as including a series of apertures (e.g., apertures 5641,apertures 5642, and the like) that function as a thermal barrier betweenadjacent portion of the assembly 5630, in other embodiments, any of thecircuit board assemblies described herein (including the assembly 5630and the assembly 7630) can include any suitable mechanism for limitingthe heat transfer between various portions of the device. For example,in some embodiments, the circuit board assembly 5630, the circuit boardassembly 7630, or any other circuit board assembly herein can includeregions that are constructed from (and/or include) a material having athermal conductivity that is lower than that of the material(s) fromwhich other portions of the circuit board assembly are constructed,similar to the heater assembly 2630 described above. For example, insome embodiments, the circuit board assembly 5630, the circuit boardassembly 7630, or any other circuit board assembly herein can includeportions that are constructed from a material having a thermalconductivity of about 0.1 W/m-K or less. In other embodiments, thecircuit board assembly 5630, the circuit board assembly 7630, or anyother circuit board assembly herein can include portions that areconstructed from a material having a thermal conductivity of about 0.05W/m-K or less. For example, in some embodiments, the circuit boardassembly 5630, the circuit board assembly 7630, or any other circuitboard assembly herein can include portions that are constructed from orinclude a rigid foam (e.g., polyurethane foam, a silicon foam, aneoprene foam, a vinyl foam, or the like).

Any of the devices and methods described herein can be utilized todetect the presence or absence of nucleic acids associated with one ormore bacterial cells in a biological sample. In some embodiments, theone or more bacterial cells are pathogens. In some embodiments, the oneor more bacterial cells are infectious. Non-limiting examples ofbacterial pathogens that can be detected include Mycobacteria (e.g., M.tuberculosis, M. bovis, M. avium, M. leprae, and M. africanum),rickettsia, mycoplasma, chlamydia, and legionella. Some examples ofbacterial infections include, but are not limited to, infections causedby Gram positive bacillus (e.g., Listeria, Bacillus such as Bacillusanthracis, Erysipelothrix species), Gram negative bacillus (e.g.,Bartonella, Brucella, Campylobacter, Enterobacter, Escherichia,Francisella, Hemophilus, Klebsiella, Morganella, Proteus, Providencia,Pseudomonas, Salmonella, Serratia, Shigella, Vibrio and Y ersiniaspecies), spirochete bacteria (e.g., Borrelia species including Borreliaburgdorferi that causes Lyme disease), anaerobic bacteria (e.g.,Actinomyces and Clostridium species), Gram positive and negative coccalbacteria, Enterococcus species, Streptococcus species, Pneumococcusspecies, Staphylococcus species, and Neisseria species. Specificexamples of infectious bacteria include, but are not limited to:Helicobacter pyloris, Legionella pneumophilia, Mycobacteriumtuberculosis, Mycobacterium avium, Mycobacterium intracellulare,Mycobacterium kansaii, Mycobacterium gordonae, Staphylococcus aureus,Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus viridans, Streptococcus faecalis,Streptococcus bovis, Streptococcus pneumoniae, Haemophilus influenzae,Bacillus antracis, Erysipelothrix rhusiopathiae, Clostridium tetani,Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida,Fusobacterium nucleatum, Streptobacillus moniliformis, Treponemapallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomycesisraelii, Acinetobacter, Bacillus, Bordetella, Borrelia, Brucella,Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium,Enterococcus, Haemophilus, Helicobacter, Mycobacterium, Mycoplasma,Stenotrophomonas, Treponema, Vibrio, Yersinia, Acinetobacter baumanii,Bordetella pertussis, Brucella abortus, Brucella canis, Brucellamelitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae,Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum,Clostridium difficile, Clostridium perfringens, Corynebacteriumdiphtheriae, Enterobacter sazakii, Enterobacter agglomerans,Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium,Escherichia coli, Francisella tularensis, Helicobacter pylori,Legionella pneumophila, Leptospira interrogans, Mycobacterium leprae,Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasmapneumoniae, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonellatyphi, Salmonella typhimurium, Salmonella enterica, Shigella sonnei,Staphylococcus epidermidis, Staphylococcus saprophyticus,Stenotrophomonas maltophilia, Vibrio cholerae, Yersinia pestis, and thelike. In some instances, the infectious bacteria is Neisseriagonorrhoeae or Chlamydia trachomatis.

Any of the devices and methods described herein can be utilized todetect the presence or absence of nucleic acids associated with one ormore viruses in a biological sample. Non-limiting examples of virusesinclude the herpes virus (e.g., human cytomegalomous virus (HCMV),herpes simplex virus I (HSV-1), herpes simplex virus 2 (HSV-2),varicella zoster virus (VZV), Epstein-Barr virus), influenza A virus andHepatitis C virus (HCV) or a picornavirus such as Coxsackievirus B3(CVB3). Other viruses may include, but are not limited to, the hepatitisB virus, HIV, poxvirus, hepadavirus, retrovirus, and RNA viruses such asflavivirus, togavirus, coronavirus, Hepatitis D virus, orthomyxovirus,paramyxovirus, rhabdovirus, bunyavirus, filo virus, Adenovirus, Humanherpesvirus, type 8, Human papillomavirus, BK virus, JC virus, Smallpox,Hepatitis B virus, Human bocavirus, Parvovirus B 19, Human astrovirus,Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus,rhinovirus, Severe acute respiratory syndrome virus, Hepatitis C virus,yellow fever virus, dengue virus, West Nile virus, Rubella virus,Hepatitis E virus, and Human immunodeficiency virus (HIV). In someembodiments, the virus is an enveloped virus. Examples of such envelopedviruses include, but are not limited to, viruses that are members of thehepadnavirus family, herpesvirus family, iridovirus family, poxvirusfamily, flavivirus family, togavirus family, retrovirus family,coronavirus family, filovirus family, rhabdovirus family, bunyavirusfamily, orthomyxovirus family, paramyxovirus family, and arenavirusfamily. Other examples include, but are not limited to, Hepadnavirushepatitis B virus (HBV), woodchuck hepatitis virus, ground squirrel(Hepadnaviridae) hepatitis virus, duck hepatitis B virus, heronhepatitis B virus, Herpesvirus herpes simplex virus (HSV) types 1 and 2,varicellazoster virus, cytomegalovirus (CMV), human cytomegalovirus(HCMV), mouse cytomegalovirus (MCMV), guinea pig cytomegalovirus(GPCMV), Epstein-Barr virus (EBV), human herpes virus 6 (HHV variants Aand B), human herpes virus 7 (HHV-7), human herpes virus 8 (HHV-8),Kaposi's sarcoma—associated herpes virus (KSHV), B virus Poxvirusvaccinia virus, variola virus, smallpox virus, monkeypox virus, cowpoxvirus, camelpox virus, ectromelia virus, mousepox virus, rabbitpoxviruses, raccoon pox viruses, molluscum contagiosum virus, orf virus,milker's nodes virus, bovin papullar stomatitis virus, sheeppox virus,goatpox virus, lumpy skin disease virus, fowlpox virus, canarypox virus,pigeonpox virus, sparrowpox virus, myxoma virus, hare fibroma virus,rabbit fibroma virus, squirrel fibroma viruses, swinepox virus, tanapoxvirus, Yabapox virus, Flavivirus dengue virus, hepatitis C virus (HCV),GB hepatitis viruses (GBV-A, GBV-B and GBV-C), West Nile virus, yellowfever virus, St. Louis encephalitis virus, Japanese encephalitis virus,Powassan virus, tick-borne encephalitis virus, Kyasanur Forest diseasevirus, Togavirus, Venezuelan equine encephalitis (VEE) virus,chikungunya virus, Ross River virus, Mayaro virus, Sindbis virus,rubella virus, Retrovirus human immunodeficiency virus (HIV) types 1 and2, human T cell leukemia virus (HTLV) types 1, 2, and 5, mouse mammarytumor virus (MMTV), Rous sarcoma virus (RSV), lentiviruses, Coronavirus,severe acute respiratory syndrome (SARS) virus, Filovirus Ebola virus,Marburg virus, Metapneumoviruses (MPV) such as human metapneumovirus(HMPV), Rhabdovirus rabies virus, vesicular stomatitis virus,Bunyavirus, Crimean-Congo hemorrhagic fever virus, Rift Valley fevervirus, La Crosse virus, Hantaan virus, Orthomyxovirus, influenza virus(types A, B, and C), Paramyxovirus, parainfluenza virus (PIV types 1, 2and 3), respiratory syncytial virus (types A and B), measles virus,mumps virus, Arenavirus, lymphocytic choriomeningitis virus, Juninvirus, Machupo virus, Guanarito virus, Lassa virus, Ampari virus, Flexalvirus, Ippy virus, Mobala virus, Mopeia virus, Latino virus, Paranavirus, Pichinde virus, Punta torn virus (PTV), Tacaribe virus andTamiami virus. In some embodiments, the virus is a non-enveloped virus,examples of which include, but are not limited to, viruses that aremembers of the parvovirus family, circovirus family, polyoma virusfamily, papillomavirus family, adenovirus family, iridovirus family,reovirus family, birnavirus family, calicivirus family, and picornavirusfamily. Specific examples include, but are not limited to, canineparvovirus, parvovirus B19, porcine circovirus type 1 and 2, BFDV (Beakand Feather Disease virus, chicken anaemia virus, Polyomavirus, simianvirus 40 (SV40), JC virus, BK virus, Budgerigar fledgling disease virus,human papillomavirus, bovine papillomavirus (BPV) type 1, cotton tailrabbit papillomavirus, human adenovirus (HAdV-A, HAdV-B, HAdV-C, HAdV-D,HAdV-E, and HAdV-F), fowl adenovirus A, bovine adenovirus D, frogadenovirus, Reovirus, human orbivirus, human coltivirus, mammalianorthoreovirus, bluetongue virus, rotavirus A, rotaviruses (groups B toG), Colorado tick fever virus, aquareovirus A, cypovirus 1, Fiji diseasevirus, rice dwarf virus, rice ragged stunt virus, idnoreovirus 1,mycoreovirus 1, Birnavirus, bursal disease virus, pancreatic necrosisvirus, Calicivirus, swine vesicular exanthema virus, rabbit hemorrhagicdisease virus, Norwalk virus, Sapporo virus, Picornavirus, humanpolioviruses (1-3), human coxsackieviruses Al-22, 24 (CA1-22 and CA24,CA23 (echovirus 9)), human coxsackieviruses (Bl-6 (CB1-6)), humanechoviruses 1-7, 9, 11-27, 29-33, vilyuish virus, simian enteroviruses1-18 (SEVI-18), porcine enteroviruses 1-11 (PEV1-11), bovineenteroviruses 1-2 (BEVI-2), hepatitis A virus, rhinoviruses,hepatoviruses, cardio viruses, aphthoviruses and echoviruses. The virusmay be phage. Examples of phages include, but are not limited to T4, TS,λ, phage, T7 phage, G4, Pl, φ6, Thermoproteus tenax virus 1, M13, MS2,Qβ, φ X174, Φ29, PZA, Φ15, BS32, B103, M2Y (M2), Nf, GA-I, FWLBc1,FWLBc2, FWLLm3, B4. The reference database may comprise sequences forphage that are pathogenic, protective, or both. In some cases, the virusis selected from a member of the Flaviviridae family (e.g., a member ofthe Flavivirus, Pestivirus, and Hepacivirus genera), which includes thehepatitis C virus, Yellow fever virus; Tick-borne viruses, such as theGadgets Gully virus, Kadam virus, Kyasanur Forest disease virus, Langatvirus, Omsk hemorrhagic fever virus, Powassan virus, Royal Farm virus,Karshi virus, tick-borne encephalitis virus, Neudoerfl virus, Sofjinvirus, Louping ill virus and the Negishi virus; seabird tick-borneviruses, such as the Meaban virus, Saumarez Reef virus, and the Tyuleniyvirus; mosquito-borne viruses, such as the Arna virus, dengue virus,Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitisvirus, Murray Valley encephalitis virus, St. Louis encephalitis virus,Usutu virus, West Nile virus, Yaounde virus, Kokobera virus, Bagazavirus, Ilheus virus, Israel turkey meningoencephalo-myelitis virus,Ntaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui virus, EdgeHill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S virus,Wesselsbron virus, yellow fever virus; and viruses with no knownarthropod vector, such as the Entebbe bat virus, Yokose virus, Apoivirus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus,San Perlita virus, Bukalasa bat virus, Carey Island virus, Dakar batvirus, Montana myotis leukoencephalitis virus, Phnom Penh bat virus, RioBravo virus, Tamana bat virus, and the Cell fusing agent virus. In somecases, the virus is selected from a member of the Arenaviridae family,which includes the Ippy virus, Lassa virus (e.g., the Josiah, LP, orGA391 strain), lymphocytic choriomeningitis virus (LCMV), Mobala virus,Mopeia virus, Amapari virus, Flexal virus, Guanarito virus, Junin virus,Latino virus, Machupo virus, Oliveros virus, Parana virus, Pichindevirus, Pirital virus, Sabia virus, Tacaribe virus, Tamiami virus,Whitewater Arroyo virus, Chapare virus, and Lujo virus. In some cases,the virus is selected from a member of the Bunyaviridae family (e.g., amember of the Hantavirus, Nairovirus, Orthobunyavirus, and Phlebovirusgenera), which includes the Hantaan virus, Sin Nombre virus, Dugbevirus, Bunyamwera virus, Rift Valley fever virus, La Crosse virus, PuntaToro virus (PTV), California encephalitis virus, and Crimean-Congohemorrhagic fever (CCHF) virus. In some cases, the virus is selectedfrom a member of the Filoviridae family, which includes the Ebola virus(e.g., the Zaire, Sudan, Ivory Coast, Reston, and Uganda strains) andthe Marburg virus (e.g., the Angola, Ci67, Musoke, Popp, Ravn and LakeVictoria strains); a member of the Togaviridae family (e.g., a member ofthe Alphavirus genus), which includes the Venezuelan equine encephalitisvirus (VEE), Eastern equine encephalitis virus (EEE), Western equineencephalitis virus (WEE), Sindbis virus, rubella virus, Semliki Forestvirus, Ross River virus, Barmah Forest virus, O′ nyong′nyong virus, andthe chikungunya virus; a member of the Poxyiridae family (e.g., a memberof the Orthopoxvirus genus), which includes the smallpox virus,monkeypox virus, and vaccinia virus; a member of the Herpesviridaefamily, which includes the herpes simplex virus (HSV; types 1, 2, and6), human herpes virus (e.g., types 7 and 8), cytomegalovirus (CMV),Epstein-Barr virus (EBV), Varicella-Zoster virus, and Kaposi's sarcomaassociated-herpesvirus (KSHV); a member of the Orthomyxoviridae family,which includes the influenza virus (A, B, and C), such as the H5N1 avianinfluenza virus or HINI swine flu; a member of the Coronaviridae family,which includes the severe acute respiratory syndrome (SARS) virus; amember of the Rhabdoviridae family, which includes the rabies virus andvesicular stomatitis virus (VSV); a member of the Paramyxoviridaefamily, which includes the human respiratory syncytial virus (RSV),Newcastle disease virus, hendravirus, nipahvirus, measles virus,rinderpest virus, canine distemper virus, Sendai virus, humanparainfluenza virus (e.g., 1, 2, 3, and 4), rhinovirus, and mumps virus;a member of the Picomaviridae family, which includes the poliovirus,human enterovirus (A, B, C, and D), hepatitis A virus, and thecoxsackievirus; a member of the Hepadnaviridae family, which includesthe hepatitis B virus; a member of the Papillamoviridae family, whichincludes the human papilloma virus; a member of the Parvoviridae family,which includes the adeno-associated virus; a member of the Astroviridaefamily, which includes the astrovirus; a member of the Polyomaviridaefamily, which includes the JC virus, BK virus, and SV40 virus; a memberof the Calciviridae family, which includes the Norwalk virus; a memberof the Reoviridae family, which includes the rotavirus; and a member ofthe Retroviridae family, which includes the human immunodeficiency virus(HIV; e.g., types I and 2), and human T-lymphotropic virus Types I andII (HTLV-1 and HTLV-2, respectively).

Any of the devices and methods described herein can be utilized todetect the presence or absence of nucleic acids associated with one ormore fungi in a biological sample. Examples of infectious fungal agentsinclude, without limitation Aspergillus, Blastomyces, Coccidioides,Cryptococcus, Histoplasma, Paracoccidioides, Sporothrix, and at leastthree genera of Zygomycetes. The above fungi, as well as many otherfungi, can cause disease in pets and companion animals. The presentteaching is inclusive of substrates that contact animals directly orindirectly. Examples of organisms that cause disease in animals includeMalassezia furfur, Epidermophyton floccosur, Trichophytonmentagrophytes, Trichophyton rubrum, Trichophyton tonsurans,Trichophyton equinum, Dermatophilus congolensis, Microsporum canis,Microsporu audouinii, Microsporum gypseum, Malassezia ovale,Pseudallescheria, Scopulariopsis, Scedosporium, and Candida albicans.Further examples of fungal infectious agent include, but are not limitedto, Aspergillus, Blastomyces dermatitidis, Candida, Coccidioidesimmitis, Cryptococcus neoformans, Histoplasma capsulatum var.capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii,Zygomycetes spp., Absidia corymbifera, Rhizomucor pusillus, or Rhizopusarrhizus.

Any of the devices and methods described herein can be utilized todetect the presence or absence of nucleic acids associated with one ormore parasites in a biological sample. Non-limiting examples ofparasites include Plasmodium, Leishmania, Babesia, Treponema, Borrelia,Trypanosoma, Toxoplasma gondii, Plasmodium falciparum, P. vivax, P.ovale, P. malariae, Trypanosoma spp., or Legionella spp. In some cases,the parasite is Trichomonas vaginalis.

1. An apparatus, comprising: a substrate including a first portion, asecond portion, and a third portion, the third portion between the firstportion and the second portion, the first portion characterized by afirst thermal conductivity, the second portion characterized by a secondthermal conductivity, the third portion characterized by a third thermalconductivity, the third thermal conductivity less than the first thermalconductivity and the second thermal conductivity; a first heatingelement coupled to the first portion of the substrate, the first heatingelement configured to produce a first thermal output; and a secondheating element coupled to the second portion of the substrate; thesecond heating element configured to produce a second thermal output,the second thermal output different from the first thermal output. 2.The apparatus of claim 1, wherein the third portion of the substratedefines an aperture.
 3. The apparatus of claim 1; wherein the thirdportion of the substrate defines a plurality of apertures that separatethe first portion of the substrate from the second portion of thesubstrate, the third portion of the substrate including a connectionportion between a first aperture from the plurality of apertures and asecond aperture from the plurality of apertures.
 4. The apparatus ofclaim 1, wherein the third thermal conductivity is less than about 0.1W/m-K.
 5. The apparatus of claim 1, wherein: the third portion of thesubstrate is constructed from a material that is different from amaterial from which at least one of the first portion of the substrateor the second portion of the substrate is constructed.
 6. (canceled) 7.The apparatus of claim 1, wherein: the first heating element is coupledto an outer surface of the first portion of the substrate; and thesecond heating element is coupled to an outer surface of the secondportion of the substrate, the outer surface of the first portion beingcoplanar with the outer surface of the second portion.
 8. The apparatusof claim 1, wherein the first heating element and the second heatingelement are constructed from a first layer bonded to the substrate, theapparatus further comprising: a second layer bonded between the firstlayer and the substrate, the second layer including a thermallyconductive portion disposed beneath the second heating element, thesecond layer including a thermally isolative portion disposed beneaththe first heating element.
 9. The apparatus of claim 1, furthercomprising: a flow member defining a flow path, the flow member coupledto the substrate such that the first heating element and the secondheating element are between the flow member and the substrate, the firstheating element and the flow member collectively configured to maintaina temperature of a first portion of the flow path at a firsttemperature, the second heating element and the flow member collectivelyconfigured to maintain a temperature of a second portion of the flowpath at a second temperature, the second temperature different from thefirst temperature.
 10. (canceled)
 11. The apparatus of claim 9, whereinthe flow member is constructed from at least one of a cyclic olefincopolymer or a graphite-based material and has a thickness of less thanabout 0.5 mm.
 12. An apparatus, comprising: a substrate defining anaperture that separates the substrate into a first portion and a secondportion; a first heating element coupled to the first portion of thesubstrate; the first heating element configured to produce a firstthermal output; and a second heating element coupled to the secondportion of the substrate, the second heating element configured toproduce a second thermal output, the second thermal output differentfrom the first thermal output.
 13. The apparatus of claim 12, wherein:the aperture is a first aperture; and the substrate defines a secondaperture and includes a connection portion between the first apertureand the second aperture.
 14. The apparatus of claim 12, wherein theaperture is a first aperture, the substrate defines a second aperturethat separates a third portion of the substrate from the first portionof the substrate, the first portion disposed between the second portionand the third portion; the apparatus further comprising: A third heatingelement coupled to the third portion of the substrate, the third heatingelement configured to produce a third thermal output, the third thermaloutput different from the first thermal output.
 15. The apparatus ofclaim 14, wherein: the first aperture is from a first plurality ofapertures, the substrate including a first connection portion betweenthe first aperture and an adjacent aperture from the first plurality ofapertures; and the second aperture is from a second plurality ofapertures, the substrate including a second connection portion betweenthe second aperture and an adjacent aperture from the second pluralityof apertures, the first connection portion offset from the secondconnection portion.
 16. The apparatus of claim 15, wherein the firstconnection portion is disposed at a first position along an axis of thesubstrate and the second connection portion is disposed at a secondposition along the axis of the substrate, the first position differentfrom the second position. 17.-25. (canceled)
 26. A method, comprising:conveying a sample into a diagnostic device including an amplificationmodule, the amplification module including a flow member and a heaterassembly, the flow member defining a flow path, the heater assemblyincluding a substrate, a first heating element, and a second heatingelement, the heater assembly coupled to the flow member such that thefirst heating element is between a first portion of the substrate and afirst portion of the flow path, and the second heating element isbetween a second portion of the substrate and a second portion of theflow path, a third portion of the substrate separating the first portionof the substrate and the second portion of the substrate, the thirdportion of the substrate characterized by a thermal conductivity that isless than a thermal conductivity of the first portion of the substrate;and actuating the diagnostic device to: supply a first current to thefirst heating element such that the first heating element maintains thefirst portion of the flow path at a first temperature; supply a secondcurrent to the second heating element such that the second heatingelement maintains the second portion of the flow path at a secondtemperature, the second temperature different from the firsttemperature; and produce a flow of the sample within the flow path. 27.The method of claim 26, wherein: the first current is supplied to thefirst heating element at a first time; and the second current issupplied to the second heating element at a second time, the second timeafter the first time.
 28. The method of claim 26, wherein: the flow ofthe sample within the flow path causes a volume of the sample to becycled between the first temperature and the second temperature suchthat a nucleic acid within the volume of the sample is amplified. 29.The method of claim 28, wherein the volume of the sample is at least 10microliters.
 30. (canceled)
 31. The method of claim 28, wherein: theflow path is a serpentine flow path having at least at least 30amplification channels the flow member and the heater assembly coupledtogether such that the volume of the sample is cycled between the firsttemperature and the second temperature when the volume of the sampleflows within each amplification channel of the serpentine flow path; andthe actuating the diagnostic device produces the flow of the samplewithin the serpentine flow path at a flow rate such that the volume ofthe sample is conveyed through the at least 30 amplification channels in15 minutes or less, the volume of the sample being at least 10microliters.
 32. (canceled)
 33. The method of claim 26, wherein thethird portion of the substrate defines an aperture. 34.-58. (canceled)